Engineered anti-il-23r antibodies

ABSTRACT

Antibodies to human IL-23R are provided, as well as uses thereof, e.g. in treatment of inflammatory, autoimmune, and proliferative disorders.

FIELD OF THE INVENTION

The present invention relates generally to antibodies specific forinterleukin-23R (IL-23R) and uses thereof. More specifically, theinvention relates to humanized antibodies that recognize human IL-23Rand modulate its activity, particularly in inflammatory, autoimmune andproliferative disorders.

BACKGROUND OF THE INVENTION

The immune system functions to protect individuals from infectiveagents, e.g., bacteria, multi-cellular organisms, and viruses, as wellas from cancers. This system includes several types of lymphoid andmyeloid cells such as monocytes, macrophages, dendritic cells (DCs),eosinophils, T cells, B cells, and neutrophils. These lymphoid andmyeloid cells often produce signaling proteins known as cytokines. Theimmune response includes inflammation, i.e., the accumulation of immunecells systemically or in a particular location of the body. In responseto an infective agent or foreign substance, immune cells secretecytokines which, in turn, modulate immune cell proliferation,development, differentiation, or migration. Immune response can producepathological consequences, e.g., when it involves excessiveinflammation, as in the autoimmune disorders. See, e.g., Abbas et al.(eds.) (2000) Cellular and Molecular Immunology, W.B. Saunders Co.,Philadelphia, Pa.; Oppenheim and Feldmann (eds.) (2001) CytokineReference, Academic Press, San Diego, Calif.; von Andrian and Mackay(2000) New Engl. J. Med. 343:1020-1034; Davidson and Diamond (2001) NewEngl. J. Med. 345:340-350.

Interleukin-12 (IL-12) is a heterodimeric molecule composed of p35 andp40 subunits. Studies have indicated that IL-12 plays a critical role inthe differentiation of naïve T cells into T-helper type 1 CD4⁺lymphocytes that secrete IFNγ. It has also been shown that IL-12 isessential for T cell dependent immune and inflammatory responses invivo. See, e.g., Cua et al. (2003) Nature 421:744-748. The IL-12receptor is composed of IL-12Rβ1 and IL-12Rβ2 subunits.

Interleukin-23 (IL-23) is a heterodimeric cytokine comprised of twosubunits, p19 which is unique to IL-23, and p40, which is shared withIL-12. The p19 subunit is structurally related to IL-6,granulocyte-colony stimulating factor (G-CSF), and the p35 subunit ofIL-12. IL-23 mediates signaling by binding to a heterodimeric receptor,comprised of IL-23R and IL-12Rβ1, which is shared by the IL-12 receptor.See Parham et al. (2000) J. Immunol. 168:5699. IL-12 receptor is acomplex of IL-12Rβ1 and IL-12Rβ2 subunits. See Presky et al. (1996)Proc. Nat'l Acad. Sci. USA 93:14002.

A number of early studies demonstrated that the consequences of agenetic deficiency in p40 (p40 knockout mouse; p40KO mouse) were moresevere than those found in a p35KO mouse. Some of these results wereeventually explained by the discovery of IL-23, and the finding that thep40KO prevents expression of not only IL-12, but also of IL-23 (see,e.g., Oppmann et al. (2000) Immunity 13:715-725; Wiekowski et al. (2001)J. Immunol. 166:7563-7570; Parham et al. (2002) J. Immunol.168:5699-708; Frucht (2002) Sci STKE 2002, E1-E3; Elkins et al. (2002)Infection Immunity 70:1936-1948).

Recent studies, through the use of p40 KO mice, have shown that blockadeof both IL-23 and IL-12 is an effective treatment for variousinflammatory and autoimmune disorders. However, the blockade of IL-12through p40 appears to have various systemic consequences such asincreased susceptibility to opportunistic microbial infections. Bowmanet al. (2006) Curr. Opin. Infect. Dis. 19:245.

IL-23R has been implicated as a critical genetic factor in theinflammatory bowel disorders Crohn's disease and ulcerative colitis.Duerr et al. (2006) Science 314:1461. A genome-wide association studyfound that the gene for IL-23R was highly associated with Crohn'sdisease, with an uncommon coding variant (Arg381Gln) conferring strongprotection against the disease. This genetic association confirms priorbiological findings (Yen et al. (2006) J. Clin. Investigation 116:1218)suggesting that IL-23 and its receptor are promising targets for newtherapeutic approaches to treating IBD.

Therapeutic antibodies may be used to block cytokine activity. The mostsignificant limitation in using antibodies as a therapeutic agent invivo is the immunogenicity of the antibodies. As most monoclonalantibodies are derived from rodents, repeated use in humans results inthe generation of an immune response against the therapeutic antibody.Such an immune response results in a loss of therapeutic efficacy at aminimum and a potential fatal anaphylactic response at a maximum.Initial efforts to reduce the immunogenicity of rodent antibodiesinvolved the production of chimeric antibodies, in which mouse variableregions were fused with human constant regions. Liu et al. (1987) Proc.Natl. Acad. Sci. USA 84:3439-43. However, mice injected with hybrids ofhuman variable regions and mouse constant regions develop a stronganti-antibody response directed against the human variable region,suggesting that the retention of the entire rodent Fv region in suchchimeric antibodies may still result in unwanted immunogenicity inpatients.

It is generally believed that complementarity determining region (CDR)loops of variable domains comprise the binding site of antibodymolecules. Therefore, the grafting of rodent CDR loops onto humanframeworks (i.e., humanization) was attempted to further minimize rodentsequences. Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988)Science 239:1534. However, CDR loop exchanges still do not uniformlyresult in an antibody with the same binding properties as the antibodyof origin. Changes in framework residues (FR), residues involved in CDRloop support, in humanized antibodies also are required to preserveantigen binding affinity. Kabat et al. (1991) J. Immunol. 147:1709.While the use of CDR grafting and framework residue preservation in anumber of humanized antibody constructs has been reported, it isdifficult to predict if a particular sequence will result in theantibody with the desired binding, and sometimes biological, properties.See, e.g., Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029,Gorman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4181, and Hodgson(1991) Biotechnology (NY) 9:421-5. Moreover, most prior studies useddifferent human sequences for animal light and heavy variable sequences,rendering the predictive nature of such studies questionable. Sequencesof known antibodies have been used or, more typically, those ofantibodies having known X-ray structures, antibodies NEW and KOL. See,e.g., Jones et al., supra; Verhoeyen et al., supra; and Gorman et al.,supra. Exact sequence information has been reported for some humanizedconstructs.

The need exists for improved methods and compositions for the treatmentof inflammatory, autoimmune, and proliferative disorders, e.g. by use ofagents that prevent IL-23 signaling through its receptor, such asantagonists of the interaction of IL-23 and the IL-23 receptor.Alternatively such agents could be used to target cells expressingIL-23R for specific ablation. Preferably, such antagonists would have ahigh affinity for the target molecule, and would be able to block theIL-23 interaction with its receptor at relatively low doses. Preferably,such methods and compositions would be highly specific for IL-23, andnot interfere with the activity of other cytokines, such as IL-12.Preferably, such methods and compositions would employ antagonistssuitable for modification for the delivery of cytotoxic payloads totarget cells, but also suitable for non-cytotoxic uses. Preferably, suchmethods and compositions would employ antibodies modified to limit theirantigenicity when administered to a subject in need thereof.

SUMMARY OF THE INVENTION

The present invention meets these needs in the art and more by providingantagonists of human IL-23R, e.g. humanized anti-human IL-23Rantibodies.

In one aspect the invention provides binding compounds, such asantibodies or fragments thereof, including humanized or chimericrecombinant antibodies, that bind to human IL-23R, comprising anantibody light chain variable domain, or antigen binding fragmentthereof, having at least one, two or three CDRs selected from the groupconsisting of SEQ ID NOs: 26-40 and 52. In one embodiment, the bindingcompound of the present invention comprises a light chain variabledomain comprising at least one CDRL1 selected from the group consistingof SEQ ID NOs: 26-30 and 52; at least one CDRL2 selected from the groupconsisting of SEQ ID NOs: 31-35; and at least one CDRL3 selected fromthe group consisting of SEQ ID NOs: 36-40.

In one embodiment, the binding compound comprises an antibody heavychain variable domain, or antigen binding fragment thereof, having atleast one, two or three CDRs selected from the group consisting of SEQID NOs: 11-25. In one embodiment, the binding compound of the presentinvention comprises a heavy chain variable domain comprising at leastone CDRH1 selected from the group consisting of SEQ ID NOs: 11-15; atleast one CDRH2 selected from the group consisting of SEQ ID NOs: 16-20;and at least one CDRH3 selected from the group consisting of SEQ ID NOs:21-25.

In other embodiments the binding compound of the present inventioncomprises a light chain variable domain and a heavy chain variabledomain, or the antigen binding fragments thereof, described in thepreceding two paragraphs.

In some embodiments, the binding compound comprises a framework region,wherein the amino acid sequence of the framework region is all orsubstantially all of a human immunoglobulin amino acid sequence.

In some embodiments the light chain and/or heavy chain variable domainscomprise a variant of one or more of the CDRs. In various embodimentsthe variant domain comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreconservatively modified amino acid residues relative to the sequence ofthe respective SEQ ID NOs. Conservative amino acid substitutions areprovided at Table 1.

In some embodiments the light chain variable domain comprises a sequenceselected from the group consisting of SEQ ID NOs: 48-49 and 53 or avariant thereof. In some embodiments the heavy chain variable domaincomprises a sequence selected from the group consisting of SEQ ID NOs:45-47. In various embodiments the variant variable domain comprises upto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 or moreconservatively modified amino acid residues relative to the sequence ofthe respective SEQ ID NOs. In yet a further embodiment, the bindingcompound comprises a light chain variable domain and a heavy chainvariable domain, or the antigen binding fragments thereof, described inthis paragraph.

In other embodiments the binding compound of the present inventioncomprises a light chain variable domain, or an antigen binding fragmentthereof, consisting essentially of a sequence selected from the groupconsisting of SEQ ID NOs: 48-49 and 53, and/or a heavy chain variabledomain, or an antigen binding fragment thereof, consisting essentiallyof a sequence selected from the group consisting of SEQ ID NOs: 45-47.

In other embodiments the binding compound of the present inventioncomprises a light chain variable domain, or an antigen binding fragmentthereof, having at least 50%, 75%, 80%, 85%, 90%, 95%, 98% or 99%sequence homology with a sequence selected from the group consisting ofSEQ ID NOs: 6-10, 48-49 and 53, and/or a heavy chain variable domain, oran antigen binding fragment thereof, having at least 50%, 75%, 80%, 85%,90%, 95%, 98% or 99% sequence homology with a sequence selected from thegroup consisting of SEQ ID NOs: 1-5 and 45-47.

In another embodiment the binding compound of the present inventioncomprises the antibody light chain variable domain of SEQ ID NOs: 49 or53 and the antibody heavy chain variable domain of SEQ ID NO: 47, or anantigen binding fragment thereof. In a further embodiment, the bindingcompound comprises the light chain of SEQ ID NOs: 51 or 54 and the heavychain of SEQ ID NO: 50, or an antigen binding fragment thereof. In yet afurther embodiment, the binding compound is encoded by the nucleic acidsequences of SEQ ID NO: 55 (heavy chain) and SEQ ID NO: 56 (lightchain). In a further embodiment, the binding compound comprises thelight chain variable domain of SEQ ID NO: 58 and the heavy chainvariable domain of SEQ ID NO: 57, or an antigen binding fragmentthereof.

In one embodiment, the invention relates to antibodies that are able toblock the binding of a binding compound of the present invention tohuman IL-23R in a cross-blocking assay. In various embodiments theantibody is able to block binding of human IL-23R to an antibodycomprising the CDR sequences of antibodies 41F11, 8B10, 3C11, 20D7 (withor without the S32T sequence variation), 20E5, as disclosed herein. Inother embodiments, the antibody is able to block binding of human IL-23Rto the antibody deposited with ATCC under accession number PTA-7800,and/or the antibody deposited under ATCC accession number PTA-7801, in across-blocking assay. In another embodiment, the invention relates tobinding compounds that are able to block IL-23R-mediated activity, suchactivities including but not limited to, binding IL-23, or mediating theproliferation or survival of T_(H)17 cells.

In some embodiments, the binding compound of the present inventioncomprises a humanized antibody comprising the CDRs, or variants thereof,selected from the CDRs of the antibodies disclosed herein, incombination with human germline light chain and heavy chain variabledomain framework sequences in place of the rodent frameworks of theparental antibodies.

In some embodiments, the binding compound of the present inventionfurther comprises a heavy chain constant region, wherein the heavy chainconstant region comprises a γ1, γ2, γ3, or γ4 human heavy chain constantregion or a variant thereof. In various embodiments the light chainconstant region comprises a lambda or a kappa human light chain constantregion.

In various embodiments the binding compounds of the present inventionare polyclonal, monoclonal, chimeric, humanized or fully humanantibodies or fragments thereof. The present invention also contemplatesthat the antigen binding fragment is an antibody fragment selected fromthe group consisting of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, and adiabody.

The present invention encompasses a method of suppressing an immuneresponse in a human subject comprising administering to a subject inneed thereof an antibody (or a antigen binding fragment thereof)specific for IL-23R in an amount effective to block IL-23 signaling. Insome embodiments, the antibody specific for IL-23R is the humanized orchimeric antibody. In further embodiments, the immune response is aninflammatory response including arthritis, psoriasis, and inflammatorybowel disease. In other embodiments, the immune response is anautoimmune response, including multiple sclerosis, uveitis, systemiclupus erythematosus and diabetes. In another embodiment, the subject hascancer and the immune response is a Th17 response.

The present invention also contemplates administering an additionalimmunosuppressive or anti-inflammatory agent. The binding compounds ofthe present invention can be in a pharmaceutical composition comprisingthe binding compound, or antigen binding fragment thereof, incombination with a pharmaceutically acceptable carrier or diluent. In afurther embodiment, the pharmaceutical composition further comprises animmunosuppressive or anti-inflammatory agent.

The present invention encompasses an isolated nucleic acid encoding thepolypeptide sequence of an antibody embodiment of the binding compoundof the present invention. The nucleic acid can be in an expressionvector operably linked to control sequences recognized by a host celltransfected with the vector. Also encompassed is a host cell comprisingthe vector, and a method of producing a polypeptide comprising culturingthe host cell under conditions wherein the nucleic acid sequence isexpressed, thereby producing the polypeptide, and recovering thepolypeptide from the host cell or medium.

In various embodiments, the invention relates to use of a bindingcompound of the present invention in the manufacture of medicaments forthe treatment of disorders including, but not limited to, inflammatorydisease, autoimmune disease, cancer, infectious disease (e.g. bacterial,mycobacterial, viral or fungal infection, including chronic infections),arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis,uveitis, systemic lupus erythematosus and diabetes. In other embodimentsthe invention relates to compositions limited by the aforementioneduses.

In other embodiments the invention relates to pharmaceuticalcompositions comprising a binding compound of the present invention fortreating disorders including, but not limited to, inflammatory disease,autoimmune disease, cancer, infectious disease (e.g. bacterial,mycobacterial, viral or fungal infection, including chronic infections),arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis,uveitis, systemic lupus erythematosus and diabetes.

In some embodiments, the binding compound or pharmaceutical compositionof the present invention induces a prolonged period of remission fromdisease symptoms in a subject, such that the dosing interval can beextended to much longer than the half-life of the binding compound inthe subject, for example in the treatment of a relapsing-remittingdisease. In various embodiments, the interval between one administrationand another is 6-, 8-, 10-, 12-, 16-, 20-, 24-, 30-weeks or longer. Inother embodiments a single administration is sufficient to permanentlyprevent relapses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows comparisons of rat (“r”), mouse (“m”) and humanized (“hu”)anti-human IL-23R antibody clone heavy chain variable domain sequences.Sequences are provided for clones r41F11, r8B10, hu8B10, r3C11, m20E5,m20D7, hu20D7-a, hu20D7-b and hu20D7-c. CDRs are indicated. Crossreferences to sequence identifiers in the Sequence Listing are providedat Table 4.

FIG. 2 shows comparisons of rat, mouse and humanized anti-human IL-23Rantibody clone light chain variable domain sequences. Sequences areprovided for clones r41F11, r8B10, hu8B10, r3C11, m20E5, m20D7,hu20D7-IV, hu20D7-II-a and hu20D7-II-b. CDRs are indicated. Crossreferences to sequence identifiers in the Sequence Listing are providedat Table 4.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise. Table 4 belowprovides a listing of sequence identifiers used in this application. Allreferences cited herein are incorporated by reference to the same extentas if each individual publication, database entry (e.g. Genbanksequences or GeneID entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.Citation of the references herein is not intended as an admission thatthe reference is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.

I. DEFINITIONS

“Proliferative activity” encompasses an activity that promotes, that isnecessary for, or that is specifically associated with, e.g., normalcell division, as well as cancer, tumors, dysplasia, celltransformation, metastasis, and angiogenesis.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding composition, or by another cell.“Treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications. “Treatment” as it applies to ahuman, veterinary, or research subject, or cell, tissue, or organ,encompasses contact of an agent with animal subject, a cell, tissue,physiological compartment, or physiological fluid. “Treatment of a cell”also encompasses situations where the agent contacts IL-23 receptor(IL-23R/IL-12Rβ1 heterodimer), e.g., in the fluid phase or colloidalphase, but also situations where the agonist or antagonist does notcontact the cell or the receptor.

As used herein, the term “antibody” refers to any form of antibody thatexhibits the desired biological activity. Thus, it is used in thebroadest sense and specifically covers monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), chimeric antibodies, humanizedantibodies, fully human antibodies, etc. so long as they exhibit thedesired biological activity.

As used herein, the terms “IL-23R binding fragment,” “binding fragmentthereof” or “antigen binding fragment thereof” encompass a fragment or aderivative of an antibody that still substantially retains itsbiological activity of binding to IL-23R. In some embodiments, anantibody or antigen binding fragment thereof of the present inventioninhibits IL-23 signaling via the IL-23 receptor, such inhibition beingreferred to herein as “IL-23R inhibitory activity.” Because antagonistsof IL-23R will have the biological activity of inhibiting IL-23signaling, such antagonists are said (interchangeably) to inhibitIL-23R, inhibit IL-23, or inhibit both IL-23/IL-23R. The term “antibodyfragment” or IL-23R binding fragment refers to a portion of a fulllength antibody, generally the antigen binding or variable regionthereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies; single-chain antibodymolecules, e.g., sc-Fv; and multispecific antibodies formed fromantibody fragments. Typically, a binding fragment or derivative retainsat least 10% of its IL-23R inhibitory activity. Preferably, a bindingfragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%,95%, 99% or 100% (or more) of its IL-23R inhibitory activity, althoughany binding fragment with sufficient affinity to exert the desiredbiological effect will be useful. It is also intended that, whenspecified, a IL-23R binding fragment can include variants havingconservative amino acid substitutions that do not substantially alterits biologic activity.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al. (1975) Nature 256: 495, or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al. (1991)Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597,for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity. U.S. Pat. No. 4,816,567;Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

As used herein, the term “single-chain Fv” or “scFv” antibody refers toantibody fragments comprising the V_(H) and V_(L) domains of antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the scFv to form thedesired structure for antigen binding. For a review of scFv, seePluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

The monoclonal antibodies herein also include camelized single domainantibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci.26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO 94/04678;WO 94/25591; U.S. Pat. No. 6,005,079). In one embodiment, the presentinvention provides single domain antibodies comprising two V_(H) domainswith modifications such that single domain antibodies are formed.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described more fully in, e.g., EP 404,097; WO93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants generally seeHolliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies contain minimalsequence derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody optionallyalso will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. The prefix “hum”,“hu” or “h” is added to antibody clone designations when necessary todistinguish humanized antibodies from parental rodent antibodies(although these same designations, depending on the context, may alsoindicate the human form of a particular protein). The humanized forms ofrodent antibodies will generally comprise the same CDR sequences of theparental rodent antibodies, although certain amino acid substitutionsmay be included to increase affinity, increase stability of thehumanized antibody, or for other reasons.

The antibodies of the present invention also include antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Suchmodification can be used to enhance or suppress various reactions of theimmune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc can alsoalter the half-life of antibodies in therapeutic antibodies. A longerhalf-life may result in less frequent dosing, with the concomitantincreased convenience and decreased use of material. See Presta (2005)J. Allergy Clin. Immunol. 116:731 at 734-35.

The antibodies of the present invention also include antibodies withintact Fc regions that provide full effector functions, e.g. antibodiesof isotype IgG1, which induce complement-dependent cytotoxicity (CDC) orantibody dependent cellular cytotoxicity (ADCC) in the a targeted cell.In some embodiments, the antibodies of the present invention areadministered to selectively deplete IL-23R-positive cells from apopulation of cells. In one embodiment, this depletion ofIL-23R-positive cells is the depletion of pathogenic Th17 cells.Depletion of such pathogenic T cell subset may result in sustainedremission when effected in subjects suffering from a relapsing/remittingautoimmune disease.

The antibodies of the present invention also include antibodiesconjugated to cytotoxic payloads, such as cytotoxic agents orradionuclides. Such antibody conjugates may be used in immunotherapy toselectively target and kill cells expressing IL-23R on their surface.Exemplary cytotoxic agents include ricin, vinca alkaloid, methotrexate,Psuedomonas exotoxin, saporin, diphtheria toxin, cisplatin, doxorubicin,abrin toxin, gelonin and pokeweed antiviral protein. Exemplaryradionuclides for use in immunotherapy with the antibodies of thepresent invention include ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹¹At, ¹⁷⁷Lu, ¹⁴³ Prand ²¹³Bi. See, e.g., U.S. Patent Application Publication No.2006/0014225.

The term “fully human antibody” refers to an antibody that compriseshuman immunoglobulin protein sequences only. A fully human antibody maycontain murine carbohydrate chains if produced in a mouse, in a mousecell, or in a hybridoma derived from a mouse cell. Similarly, “mouseantibody” or “rat antibody” refer to an antibody that comprises onlymouse or rat immunoglobulin sequences, respectively. A fully humanantibody may be generated in a human being, in a transgenic animalhaving human immunoglobulin germline sequences, by phage display orother molecular biological methods.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34(CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variabledomain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) inthe heavy chain variable domain (Kabat et al. (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.) and/or those residues froma “hypervariable loop” (i.e. residues 26-32 (L1), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987)J. Mol. Biol. 196: 901-917). As used herein, the term “framework” or“FR” residues refers to those variable domain residues other than thehypervariable region residues defined herein as CDR residues. Theresidue numbering above relates to the Kabat numbering system and doesnot necessarily correspond in detail to the sequence numbering in theaccompanying Sequence Listing. Sequence variants in both the CDR andframework regions are contemplated, and may be represented as “XbbZ”, inwhich amino acid “X” at position “bb” is replaced by amino acid “Z”, andwherein X and Z are in either triple- or single-letter amino acid code,and position number “bb” is typically defined with respect to thenumbering of a specific sequence disclosed in the Sequence Listing.

“Binding compound” refers to a molecule, small molecule, macromolecule,polypeptide, antibody or fragment or analogue thereof, or solublereceptor, capable of binding to a target. “Binding compound” also mayrefer to a complex of molecules, e.g., a non-covalent complex, to anionized molecule, and to a covalently or non-covalently modifiedmolecule, e.g., modified by phosphorylation, acylation, cross-linking,cyclization, or limited cleavage, that is capable of binding to atarget. When used with reference to antibodies, the term “bindingcompound” refers to both antibodies and antigen binding fragmentsthereof. “Binding” refers to an association of the binding compound witha target where the association results in reduction in the normalBrownian motion of the binding compound, in cases where the bindingcompound can be dissolved or suspended in solution. “Bindingcomposition” refers to a molecule, e.g. a binding compound, incombination with a stabilizer, excipient, salt, buffer, solvent, oradditive, capable of binding to a target.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids are known to those of skill in this artand may often be made even in essential regions of the polypeptidewithout altering the biological activity of the resulting molecule. Suchexemplary substitutions are preferably made in accordance with those setforth in Table 1 as follows:

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys, His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide may notsubstantially alter biological activity. See, e.g., Watson et al. (1987)Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224(4th Edition).

The phrase “consists essentially of,” or variations such as “consistessentially of” or “consisting essentially of,” as used throughout thespecification and claims, indicate the inclusion of any recited elementsor group of elements, and the optional inclusion of other elements, ofsimilar or different nature than the recited elements, that do notmaterially change the basic or novel properties of the specified dosageregimen, method, or composition. As a non-limiting example, a bindingcompound that consists essentially of a recited amino acid sequence mayalso include one or more amino acids, including substitutions of one ormore amino acid residues, that do not materially affect the propertiesof the binding compound.

“Effective amount” encompasses an amount sufficient to ameliorate orprevent a symptom or sign of the medical condition. Such an effectiveamount need not necessarily completely ameliorate or prevent suchsymptom or sign. Effective amount also means an amount sufficient toallow or facilitate diagnosis. An effective amount for a particularpatient or veterinary subject may vary depending on factors such as thecondition being treated, the overall health of the patient, the methodroute and dose of administration and the severity of side affects. See,e.g., U.S. Pat. No. 5,888,530. An effective amount can be the maximaldose or dosing protocol that avoids significant side effects or toxiceffects. The effect will result in an improvement of a diagnosticmeasure or parameter by at least 5%, usually by at least 10%, moreusually at least 20%, most usually at least 30%, preferably at least40%, more preferably at least 50%, most preferably at least 60%, ideallyat least 70%, more ideally at least 80%, and most ideally at least 90%,where 100% is defined as the diagnostic parameter shown by a normalsubject. See, e.g., Maynard et al. (1996) A Handbook of SOPs for GoodClinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK.

“Immune condition” or “immune disorder” encompasses, e.g., pathologicalinflammation, an inflammatory disorder, and an autoimmune disorder ordisease. “Immune condition” also refers to infections, persistentinfections, and proliferative conditions, such as cancer, tumors, andangiogenesis, including infections, tumors, and cancers that resisteradication by the immune system. “Cancerous condition” includes, e.g.,cancer, cancer cells, tumors, angiogenesis, and precancerous conditionssuch as dysplasia.

“Inflammatory disorder” means a disorder or pathological condition wherethe pathology results, in whole or in part, from, e.g., a change innumber, change in rate of migration, or change in activation, of cellsof the immune system. Cells of the immune system include, e.g., T cells,B cells, monocytes or macrophages, antigen presenting cells (APCs),dendritic cells, microglia, NK cells, NKT cells, neutrophils,eosinophils, mast cells, or any other cell specifically associated withthe immunology, for example, cytokine-producing endothelial orepithelial cells.

An “IL-17-producing cell” means a T cell that is not a classicalTH1-type T cell or classical TH2-type T cell, referred to as T_(H)17cells. T_(H)17 cells are discussed in greater detail at Cua andKastelein (2006) Nat. Immunol. 7:557-559; Tato and O'Shea (2006) Nature441:166-168; Iwakura and Ishigame (2006) J. Clin. Invest. 116:1218-1222.“IL-17-producing cell” also means a T cell that expresses a gene orpolypeptide of Table 10B of U.S. Patent Application Publication No.2004/0219150 (e.g., mitogen responsive P-protein; chemokine ligand 2;interleukin-17 (IL-17); transcription factor RAR related; and/orsuppressor of cytokine signaling 3), where expression with treatment byan IL-23 agonist is greater than treatment with an IL-12 agonist, where“greater than” is defined as follows. Expression with an IL-23 agonistis ordinarily at least 5-fold greater, typically at least 10-foldgreater, more typically at least 15-fold greater, most typically atleast 20-fold greater, preferably at least 25-fold greater, and mostpreferably at least 30-fold greater, than with IL-12 treatment.Expression can be measured, e.g., with treatment of a population ofsubstantially pure IL-17 producing cells. A Th17 response is an immuneresponse in which the activity and/or proliferation of Th17 cells areenhanced, typically coupled with a repressed Th1 response.

Moreover, “IL-17-producing cell” includes a progenitor or precursor cellthat is committed, in a pathway of cell development or celldifferentiation, to differentiating into an IL-17-producing cell, asdefined above. A progenitor or precursor cell to the IL-17 producingcell can be found in a draining lymph node (DLN). Additionally,“IL-17-producing cell” encompasses an IL-17-producing cell, as definedabove, that has been, e.g., activated, e.g., by a phorbol ester,ionophore, and/or carcinogen, further differentiated, stored, frozen,desiccated, inactivated, partially degraded, e.g., by apoptosis,proteolysis, or lipid oxidation, or modified, e.g., by recombinanttechnology.

As used herein, the term “isolated nucleic acid molecule” refers to anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the antibody nucleic acid. An isolated nucleicacid molecule is other than in the form or setting in which it is foundin nature. Isolated nucleic acid molecules therefore are distinguishedfrom the nucleic acid molecule as it exists in natural cells. However,an isolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the antibody where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

The expression “control sequences” refers to DNA sequences involved inthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure or technique in which minute amounts of a specific piece ofnucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.Pat. No. 4,683,195. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers can coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al. (1987) Cold Spring HarborSymp. Quant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (StocktonPress, N.Y.) As used herein, PCR is considered to be one, but not theonly, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a knownnucleic acid as a primer and a nucleic acid polymerase to amplify orgenerate a specific piece of nucleic acid.

As used herein, the term “germline sequence” refers to a sequence ofunrearranged immunoglobulin DNA sequences, including rodent (e.g. mouse)and human germline sequences. Any suitable source of unrearrangedimmunoglobulin DNA may be used. Human germline sequences may beobtained, for example, from JOINSOLVER® germline databases on thewebsite for the National Institute of Arthritis and Musculoskeletal andSkin Diseases of the United States National Institutes of Health. Mousegermline sequences may be obtained, for example, as described inGiudicelli et al. (2005) Nucleic Acids Res. 33:D256-D261.

To examine the extent of inhibition of IL-23/IL-23R activity, forexample, samples or assays comprising a given, e.g., protein, gene,cell, or organism, are treated with a potential activating or inhibitingagent and are compared to control samples without the agent. Controlsamples, i.e., not treated with agent, are assigned a relative activityvalue of 100% Inhibition is achieved when the activity value relative tothe control is about 90% or less, typically 85% or less, more typically80% or less, most typically 75% or less, generally 70% or less, moregenerally 65% or less, most generally 60% or less, typically 55% orless, usually 50% or less, more usually 45% or less, most usually 40% orless, preferably 35% or less, more preferably 30% or less, still morepreferably 25% or less, and most preferably less than 20%. Activation isachieved when the activity value relative to the control is about 110%,generally at least 120%, more generally at least 140%, more generally atleast 160%, often at least 180%, more often at least 2-fold, most oftenat least 2.5-fold, usually at least 5-fold, more usually at least10-fold, preferably at least 20-fold, more preferably at least 40-fold,and most preferably over 40-fold higher.

Endpoints in activation or inhibition can be monitored as follows.Activation, inhibition, and response to treatment, e.g., of a cell,physiological fluid, tissue, organ, and animal or human subject, can bemonitored by an endpoint. The endpoint may comprise a predeterminedquantity or percentage of, e.g., an indicia of inflammation,oncogenicity, or cell degranulation or secretion, such as the release ofa cytokine, toxic oxygen, or a protease. The endpoint may comprise,e.g., a predetermined quantity of ion flux or transport; cell migration;cell adhesion; cell proliferation; potential for metastasis; celldifferentiation; and change in phenotype, e.g., change in expression ofgene relating to inflammation, apoptosis, transformation, cell cycle, ormetastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158;Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme et al. (2003)Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med. Clin.North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev. GenomicsHum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243;Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

An endpoint of inhibition is generally 75% of the control or less,preferably 50% of the control or less, more preferably 25% of thecontrol or less, and most preferably 10% of the control or less.Generally, an endpoint of activation is at least 150% the control,preferably at least two times the control, more preferably at least fourtimes the control, and most preferably at least 10 times the control.

“Small molecule” is defined as a molecule with a molecular weight thatis less than 10 kDa, typically less than 2 kDa, and preferably less than1 kDa. Small molecules include, but are not limited to, inorganicmolecules, organic molecules, organic molecules containing an inorganiccomponent, molecules comprising a radioactive atom, synthetic molecules,peptide mimetics, and antibody mimetics. As a therapeutic, a smallmolecule may be more permeable to cells, less susceptible todegradation, and less apt to elicit an immune response than largemolecules. Small molecules, such as peptide mimetics of antibodies andcytokines, as well as small molecule toxins are described. See, e.g.,Casset et al. (2003) Biochem. Biophys. Res. Commun. 307:198-205;Muyldermans (2001) J. Biotechnol. 74:277-302; L1 (2000) Nat. Biotechnol.18:1251-1256; Apostolopoulos et al. (2002) Curr. Med. Chem. 9:411-420;Monfardini et al. (2002) Curr. Pharm. Des. 8:2185-2199; Domingues et al.(1999) Nat. Struct. Biol. 6:652-656; Sato and Sone (2003) Biochem. J.371:603-608; U.S. Pat. No. 6,326,482.

“Specifically” or “selectively” binds, when referring to aligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction that is determinative of the presence of the protein ina heterogeneous population of proteins and other biologics. Thus, underdesignated conditions, a specified ligand binds to a particular receptorand does not bind in a significant amount to other proteins present inthe sample. As used herein, an antibody is said to bind specifically toa polypeptide comprising a given sequence (in this case IL-23R) if itbinds to polypeptides comprising the sequence of IL-23R but does notbind to proteins lacking the sequence of IL-23R. For example, anantibody that specifically binds to a polypeptide comprising IL-23R maybind to a FLAG®-tagged form of IL-23R but will not bind to otherFLAG®-tagged proteins.

The antibody, or binding composition derived from the antigen-bindingsite of an antibody, of the contemplated method binds to its antigenwith an affinity that is at least two fold greater, preferably at leastten times greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with unrelatedantigens. In a preferred embodiment the antibody will have an affinitythat is greater than about 10⁹ liters/mol, as determined, e.g., byScatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107:220-239.

As used herein, the term “immunomodulatory agent” refers to natural orsynthetic agents that suppress or modulate an immune response. Theimmune response can be a humoral or cellular response. Immunomodulatoryagents encompass immunosuppressive or anti-inflammatory agents.

“Immunosuppressive agents,” “immunosuppressive drugs,” or“immunosuppressants” as used herein are therapeutics that are used inimmunosuppressive therapy to inhibit or prevent activity of the immunesystem. Clinically they are used to prevent the rejection oftransplanted organs and tissues (e.g. bone marrow, heart, kidney,liver), and/or in the treatment of autoimmune diseases or diseases thatare most likely of autoimmune origin (e.g. rheumatoid arthritis,myasthenia gravis, systemic lupus erythematosus, ulcerative colitis,multiple sclerosis). Immunosuppressive drugs can be classified into fourgroups: glucocorticoids cytostatics; antibodies (including BiologicalResponse Modifiers or DMARDs); drugs acting on immunophilins; otherdrugs, including known chemotherapeutic agents used in the treatment ofproliferative disorders. For multiple sclerosis, in particular, theantibodies of the present invention can be administered in conjunctionwith a new class of myelin binding protein-like therapeutics, known ascopaxones.

“Anti-inflammatory agents” or “anti-inflammatory drugs”, is used torepresent both steroidal and non-steroidal therapeutics. Steroids, alsoknown as corticosteroids, are drugs that closely resemble cortisol, ahormone produced naturally by adrenal glands. Steroids are used as themain treatment for certain inflammatory conditions, such as: Systemicvasculitis (inflammation of blood vessels); and Myositis (inflammationof muscle). Steroids might also be used selectively to treatinflammatory conditions such as: rheumatoid arthritis (chronicinflammatory arthritis occurring in joints on both sides of the body);systemic lupus erythematosus (a generalized disease caused by abnormalimmune system function); Sjögren's syndrome (chronic disorder thatcauses dry eyes and a dry mouth).

Non-steroidal anti-inflammatory drugs, usually abbreviated to NSAIDs,are drugs with analgesic, antipyretic and anti-inflammatory effects—theyreduce pain, fever and inflammation. The term “non-steroidal” is used todistinguish these drugs from steroids, which (amongst a broad range ofother effects) have a similar eicosanoid-depressing, anti-inflammatoryaction. NSAIDs are generally indicated for the symptomatic relief of thefollowing conditions: rheumatoid arthritis; osteoarthritis; inflammatoryarthropathies (e.g. ankylosing spondylitis, psoriatic arthritis,Reiter's syndrome); acute gout; dysmenorrhoea; metastatic bone pain;headache and migraine; postoperative pain; mild-to-moderate pain due toinflammation and tissue injury; pyrexia; and renal colic. NSAIDs includesalicylates, arlyalknoic acids, 2-arylpropionic acids (profens),N-arylanthranilic acids (fenamic acids), oxicams, coxibs, andsulphonanilides.

II. GENERAL

The present invention provides engineered anti-IL-23R antibodies anduses thereof to treat inflammatory, autoimmune, and proliferativedisorders.

A number of cytokines have a role in the pathology or repair ofneurological disorders. IL-6, IL-17, interferon-gamma (IFNgamma, IFN-γ),and granulocyte colony-stimulating factor (GM-CSF) have been associatedwith multiple sclerosis. Matusevicius et al. (1999) Multiple Sclerosis5:101-104; Lock et al. (2002) Nature Med. 8:500-508. IL-1alpha,IL-1beta, and transforming growth factor-beta 1 (TGF-beta1) play a rolein ALS, Parkinson's disease, and Alzheimer's disease. Hoozemans et al.(2001) Exp. Gerontol. 36:559-570; Griffin and Mrak (2002) J. LeukocyteBiol. 72:233-238; Ilzecka et al. (2002) Cytokine 20:239-243. TNF-alpha,IL-1beta, IL-6, IL-8, interferon-gamma, and IL-17 appear to modulateresponse to brain ischemia. See, e.g., Kostulas et al. (1999) Stroke30:2174-2179; Li et al. (2001) J. Neuroimmunol. 116:5-14. Vascularendothelial cell growth factor (VEGF) is associated with ALS. Clevelandand Rothstein (2001) Nature 2:806-819.

Inflammatory bowel disorders, e.g., Crohn's disease, ulcerative colitis,celiac disease, and irritable bowel syndrome, are mediated by cells ofthe immune system and by cytokines. For example, Crohn's disease isassociated with increased IL-12 and IFNγ, while ulcerative colitis isassociated with increased IL-5, IL-13, and transforming growthfactor-beta (TGFbeta). IL-17 expression may also increase in Crohn'sdisease and ulcerative colitis. See, e.g., Podolsky (2002) New Engl. J.Med. 347:417-429; Bouma and Strober (2003) Nat. Rev. Immunol. 3:521-533;Bhan et al. (1999) Immunol. Rev. 169:195-207; Hanauer (1996) New Engl.J. Med. 334:841-848; Green (2003) The Lancet 362:383-391; McManus (2003)New Engl. J. Med. 348:2573-2574; Horwitz and Fisher (2001) New Engl. J.Med. 344:1846-1850; Andoh et al. (2002) Int. J. Mol. Med. 10:631-634;Nielsen et al. (2003) Scand. J. Gastroenterol. 38:180-185; Fujino et al.(2003) Gut 52:65-70.

IL-23 receptor is a heterodimeric complex of IL-23R and IL-12Rβ1subunits. See Parham et al. (2000) J. Immunol. 168:5699. IL-12 receptoris a complex of IL-12Rβ1 and IL-12Rβ2 subunits. See Presky et al. (1996)Proc. Nat'l Acad. Sci. USA 93:14002. IL-23R has been implicated as acritical genetic factor in the inflammatory bowel disorders Crohn'sdisease and ulcerative colitis. Duerr et al. (2006) Science 314:1461. Agenome-wide association study found that the gene for IL-23R was highlyassociated with Crohn's disease, with an uncommon coding variant(Arg381Gln) conferring strong protection against the disease. Thisgenetic association confirms prior biological findings (Yen et al.(2006) J. Clin. Investigation 116:1218) suggesting that IL-23 and itsreceptor (including IL-23R) are promising targets for new therapeuticapproached to treating IBD.

Inflammatory diseases of the skin, joints, CNS, as well as proliferativedisorders elicit similar immune responses, thus IL-23/IL-23R blockadeshould provide inhibition of these immune mediated inflammatorydisorders, without comprising the host ability to fight systemicinfections. Antagonizing IL-23/IL-23R should relieve the inflammationassociated with inflammatory bowel disease, Crohn's disease, ulcerativecolitis, rheumatoid arthritis, psoriatic arthritis, psoriasis,ankylosing spondylitis and atopic dermatitis. Use of IL-23/IL-23Rinhibitors will also provide inhibition of proliferative disorders,e.g., cancer and autoimmune disorders e.g., multiple sclerosis, type Idiabetes, and SLE. Descriptions of IL-23 in these various disorders canbe found in the following published PCT applications: WO 04/081190; WO04/071517; WO 00/53631; and WO 01/18051. IL-23/IL-23R inhibitors mayalso find use in treatment of infections, including chronic infections,such as bacterial, mycobacterial, viral and fungal infections.

The p19 subunit of IL-23 is a member of the IL-6 family of helicalcytokines The p19 subunit interacts with three cytokine receptorsubunits to form the competent signaling complex. When expressed in acell, the p19 subunit first forms a complex with the p40 subunit, whichit shares with IL-12. As noted above, the p19p40 complex is secretedfrom the cell as a heterodimeric protein and is called IL-23 (see, e.g.,Oppmann et al., supra).

The cellular receptor complex required to transduce the IL-23 signalconsists of two members of the tall signaling receptor subunits of theIL-6/IL-12 family of cytokines, the IL-23-specific IL-23R (see, e.g.,Parham et al. supra) and the IL-12Rβ1 subunit, which is shared withIL-12. The amino acid sequence for human IL-23R is found at GenBankAccession No: AAM44229 (SEQ ID NO: 41), and an mRNA sequence is found atGenBank Accession No: NM_(—)144701. The human IL-23R gene is describedat NCBI GeneID No. 149233. The amino acid sequence for mouse (musmusculus) IL-23R is found at GenBank Accession No: AAM44230, andprovided at SEQ ID NO: 42. Amino acid residues 1-23 of both human andmouse IL-23R sequences represent signal sequences that are not presentin the mature forms of IL-23R. The amino acid sequences for human andmouse (mus musculus) IL-12Rβ1 are found at GenBank Accession Nos:NP_(—)005526 and Q60837, and provided at SEQ ID NOs: 43 and 44,respectively. Amino acid residues 1-24 and 1-19 represent signalsequences for human and mouse IL-12Rβ1, respectively.

Comparison of the natural roles of IL-12 and IL-23 suggest thattargeting IL-23 for inhibition will cause fewer adverse side-effectswhen compared with inhibition of IL-12, or inhibition of both IL-23 andIL-12. Bowman et al. (2006) Curr. Opin. Infect. Dis. 19:245. While IL-12is critical to mounting a systemic Th1-mediated immune responses, IL-23(along with IL-6 and TNF-α) is thought to be responsible for promotionand maintenance of Th-17 cells. Such Th-17 cells are believed to beinvolved in responses to catastrophic injury, such as breach of themucosal barrier of the lung or gut, and the resulting exposure to thedeadly pathogens K. pneumoniae and C. rodentium. Such catastrophicinjuries would almost certainly require an immediate immune response inthe form of massive neutrophil influx. See Cua and Kastelein (2006)Nature Immunology 7:557. Because such catastrophic injuries andinfections are relatively rare in modern society, and can be treatedwith antibiotics if they do occur, this Th-17 “nuclear option” may notbe as critical to survival as it was earlier in human evolution. Thissuggests that disruption of IL-23/IL-23 receptor signaling may have arelatively minor side effect profile, since its natural activity is oflittle importance in modern society. See McKenzie et al. (2006) TrendsImmunol. 27:17.

The distinct subunit compositions of IL-12 receptor and IL-23 receptormake it possible to design therapy that targets only IL-23 receptor butnot IL-12 receptor. Compounds that bind to and inhibit the activity ofIL-23p19 or IL-23R, either in isolation of as components of theirrespective heterodimeric complexes, will inhibit IL-23 but not IL-12.There may also be compounds that are capable of binding to IL-12p40 whenpresent in IL-23 but not in IL-12, or compounds that bind to and inhibitIL-12Rβ1 when present in the IL-23 receptor but not in IL-12 receptor.Such specific binding agents will also inhibit IL-23 activity but notIL-12 activity. IL-23/IL-23R specific agents would be expected to besafer (i.e. have a lower side effect profile) than agents that alsoinhibit IL-12.

Much of the early work on inhibition of IL-12 involved inhibition ofIL-12p40. It has been subsequently realized that these experimentsinvolved not only inhibition of IL-12 but also inhibition of IL-23, andthat in fact the effects in many of these experiments were the result ofinhibition of IL-23. Many disorders once thought to be caused by apathogenic Th1 response, which could be ameliorated by inhibition ofIL-12, have been shown instead to be caused by a Th17 response, which isameliorated by inhibition of IL-23. Yen et al. (2006) J. Clin. Invest.116:1310; Iwakura and Ishingame (2006) J. Clin. Invest. 116:1218.

IL-23 receptor, and specifically IL-23R, represents an attractive targetfor therapeutic intervention. IL-23R is expressed on the surface ofT_(H)17 cells, and the antibodies of the present invention may be usedto deliver a cytotoxic payload specifically to that T-cell subset. Suchcell-specific immunotherapy is particularly suited to treatment ofdisorders in which T_(H)17 cells migrate from a tissue in which they areinduced (e.g. the gut, lung or skin) into another tissue, such as CNS,since the cells could be targeted and killed prior to infiltrating thenew target tissue and inducing disease (e.g. MS in the CNS). See Chen etal. (2006) J. Clin. Invest. 116:1317.

In other embodiments, however, it may be preferable to avoid cytotoxiceffects in cells expressing IL-23R, but instead to simply block IL-23signaling. In the case of treatment of tumors (see, e.g., WO2004/081190), for example, it may be preferable to block IL-23 signalingin Th0 cells, and thus block Th17 formation, without inducing cellkilling. The Th0 cells will then be available for production cells ofthe Th1 lineage, induced by IL-12, which can then engage in productiveimmune surveillance of the tumor cells with beneficial effect. Thecompositions and methods of the present invention encompass bothscenarios by providing binding compounds (antibodies) that may or maynot enhance killing of IL-23R expressing cells, depending on which isthe preferred approach for the specific therapeutic use. In someembodiments, antibodies or fragments thereof lacking effector functionmay be used, such as IgG antibodies other than subclass IgG₁, or Fab orother antibody fragments.

Targeting IL-23R has advantages over targeting IL-12Rβ1, the othersubunit of the heterodimeric IL-23 receptor, since inhibition of IL-23Rwill disrupt IL-23 signaling but not disrupt IL-12 signaling Inhibitionof IL-12Rβ1 would disrupt signaling of both IL-23 and IL-12. In generalprinciple a more narrowly targeted therapy is preferable, and in thisspecific case there are good reasons to expect that disruption of IL-12would reduce the subject's ability to resist some infectious diseasesand to maintain tumor surveillance.

Targeting IL-23R has advantages over targeting IL-23p19 despite the factthat the two therapeutic approaches both act by disrupting IL-23signaling through its receptor. IL-23R is present at significantly lowerlevels than IL-23p19, and the amount of anti-IL-23R antibody necessaryto block IL-23R would be expected to be correspondingly lower than wouldbe required to block the activity of p19 in a subject. This reduction inthe amount of antibody may provide several advantages, such as reducingthe expense of treatment, decreasing the concentration and/or volume ofthe drug to be delivered, and facilitating less frequent administration.In addition, targeting of IL-23p19 may alter the circulating level ofIL-12p40, which is a subunit of both IL-23 and IL-12, and also appearsto exist in a p40 homodimer form. Because the potential biologicaleffects of alterations in the levels of IL-12 and/or IL-12p40 homodimerare not fully understood, it would be preferable to block IL-23signaling in a way that doesn't involve targeting the soluble cytokine(IL-23), but instead targets the IL-23-specific receptor subunit IL-23R.

III. GENERATION OF IL-23R SPECIFIC ANTIBODIES

Any suitable method for generating monoclonal antibodies may be used.For example, a recipient may be immunized with IL-23R or a fragmentthereof. Any suitable method of immunization can be used. Such methodscan include adjuvants, other immunostimulants, repeated boosterimmunizations, and the use of one or more immunization routes. Anysuitable source of IL-23R can be used as the immunogen for thegeneration of the non-human antibody of the compositions and methodsdisclosed herein. Such forms include, but are not limited whole protein,peptide(s), and epitopes generated through recombinant, synthetic,chemical or enzymatic degradation means known in the art. In preferredembodiments the immunogen comprises the extracellular portion of IL-23R.The domain structure of IL-23R is discussed in U.S. Pat. No. 6,756,481,in which the extracellular domain is predicted to comprise amino acids1-328 of the mature form of human IL-23R, corresponding to residues24-351 of SEQ ID NO: 41 of the present application. See also WO2004/052157. The extracellular domain may be defined differentlydepending on the proposed purpose, such as design of an immunogenicpolypeptide construct.

Any form of the antigen can be used to generate the antibody that issufficient to generate a biologically active antibody. Thus, theeliciting antigen may be a single epitope, multiple epitopes, or theentire protein alone or in combination with one or more immunogenicityenhancing agents known in the art. The eliciting antigen may be anisolated full-length protein, a cell surface protein (e.g., immunizingwith cells transfected with at least a portion of the antigen), or asoluble protein (e.g., immunizing with only the extracellular domainportion of the protein). The antigen may be produced in a geneticallymodified cell. The DNA encoding the antigen may genomic or non-genomic(e.g., cDNA) and encodes at least a portion of the extracellular domain.As used herein, the term “portion” refers to the minimal number of aminoacids or nucleic acids, as appropriate, to constitute an immunogenicepitope of the antigen of interest. Any genetic vectors suitable fortransformation of the cells of interest may be employed, including butnot limited to adenoviral vectors, plasmids, and non-viral vectors, suchas cationic lipids.

Any suitable method can be used to elicit an antibody with the desiredbiologic properties to inhibit IL-23/IL-23R. It is desirable to preparemonoclonal antibodies (mAbs) from various mammalian hosts, such as mice,rats, other rodents, humans, other primates, etc. Description oftechniques for preparing such monoclonal antibodies may be found in,e.g., Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding(1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) AcademicPress, New York, N.Y. Thus, monoclonal antibodies may be obtained by avariety of techniques familiar to researchers skilled in the art.Typically, spleen cells from an animal immunized with a desired antigenare immortalized, commonly by fusion with a myeloma cell. See Kohler andMilstein (1976) Eur. J. Immunol. 6:511-519. Alternative methods ofimmortalization include transformation with Epstein Barr Virus,oncogenes, or retroviruses, or other methods known in the art. See,e.g., Doyle et al. (eds. 1994 and periodic supplements) CELL AND TISSUECULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y.Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monoclonal antibodies produced by such cellsmay be enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. Alternatively, one may isolateDNA sequences that encode a monoclonal antibody or a antigen bindingfragment thereof by screening a DNA library from human B cellsaccording, e.g., to the general protocol outlined by Huse et al. (1989)Science 246:1275-1281.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse et al. supra; and Ward etal. (1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance that provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al. (1989)Proc. Nat'l Acad. Sci. USA 86:10029-10033; or made in transgenic mice,see Mendez et al. (1997) Nature Genetics 15:146-156. See also Abgenixand Medarex technologies.

Antibodies or binding compositions against predetermined fragments ofIL-23R can be raised by immunization of animals with conjugates of thepolypeptide, fragments, peptides, or epitopes with carrier proteins.Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies can be screened for binding to normal ordefective IL-23R. These monoclonal antibodies will usually bind with atleast a K_(d) of about 1 μM, more usually at least about 300 nM, 30 nM,10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM, 10 pM, 1 pM or better, usuallydetermined by ELISA or BIAcore® label-free interaction analysis system.Suitable non-human antibodies may also be identified using the biologicassays described in Examples 5, 6 and 7, below.

Hybridomas expressing antibodies 8B10 (rat IgG2a kappa) and 20D7 (mouseIgG1 kappa) were deposited pursuant to the Budapest Treaty with AmericanType Culture Collection (ATCC—Manassas, Va., USA) on Aug. 17, 2006 underAccession Numbers PTA-7800 and PTA-7801, respectively.

IV. HUMANIZATION OF IL-23R SPECIFIC ANTIBODIES

Any suitable non-human antibody can be used as a source for thehypervariable region. Sources for non-human antibodies include, but arenot limited to, murine (e.g. Mus musculus), rat (e.g. Rattusnorvegicus), Lagomorphs (including rabbits), bovine, and primates. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primate havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance of the desired biological activity. For furtherdetails, see Jones et al. (1986) Nature 321:522-525; Reichmann et al.(1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol.2:593-596.

Methods for recombinantly engineering antibodies have been described,e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S.Pat. No. 4,816,567), Law et al. (European Patent Application PublicationNo. EP438310A1) and Winter (European Patent Application Publication No.EP239400B1).

Amino acid sequence variants of humanized anti-IL-23R antibody areprepared by introducing appropriate nucleotide changes into thehumanized anti-IL-23R antibody DNA, or by peptide synthesis. Suchvariants include, for example, deletions from, and/or insertions into,and/or substitutions of, residues within the amino acid sequences shownfor the humanized anti-IL-23R antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe humanized anti-IL-23R antibody, such as changing the number orposition of glycosylation sites.

A useful method for identification of certain residues or regions of thehumanized anti-IL-23R antibody polypeptide that are preferred locationsfor mutagenesis is called “alanine scanning mutagenesis,” as describedby Cunningham and Wells (1989) Science 244: 1081-1085. Here, a residueor group of target residues are identified (e.g., charged residues suchas Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with IL-23R antigen. The amino acidresidues demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, Ala scanning or random mutagenesis isconducted at the target codon or region and the expressed humanizedanti-IL-23R antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includehumanized anti-IL-23R antibody with an N-terminal methionyl residue orthe antibody fused to an epitope tag. Other insertional variants of thehumanized anti-IL-23R antibody molecule include the fusion to the N- orC-terminus of humanized anti-IL-23R antibody of an enzyme or apolypeptide that increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the humanizedanti-IL-23R antibody molecule removed and a different residue insertedin its place. The sites of greatest interest for substitutionalmutagenesis include the hypervariable loops, but FR alterations are alsocontemplated.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Yet another type of amino acid variant is the substitution of residuesto provide for greater chemical stability of the final humanizedantibody. For example, an asparagine (N) residue may be changed toreduce the potential for formation of isoaspartate at any NG sequenceswithin a rodent CDR. A similar problem may occur at a DG sequence.Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartateformation may debilitate or completely abrogate binding of an antibodyto its target antigen. Presta (2005) J. Allergy Clin. Immunol. 116:731at 734. In one embodiment, the asparagine is changed to glutamine (Q).It may also be desirable to alter an amino acid adjacent to anasparagine (N) or glutamine (Q) residue to reduce the likelihood ofdeamidation, which occurs at greater rates when small amino acids occuradjacent to asparagine or glutamine. Bischoff & Kolbe (1994) J.Chromatog. 662:261. In addition, methionine residues in rodent CDRs maybe changed to reduce the possibility that the methionine sulfur wouldoxidize, which could reduce antigen binding affinity and also contributeto molecular heterogeneity in the final antibody preparation. Id. In oneembodiment, the methionine is changed to alanine (A). Antibodies withsuch substitutions are subsequently screened to ensure that thesubstitutions do not decrease IL-23R binding affinity to unacceptablelevels.

Nucleic acid molecules encoding amino acid sequence variants ofhumanized IL-23R specific antibody are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofhumanized anti-IL-23R antibody.

Ordinarily, amino acid sequence variants of the humanized anti-IL-23Rantibody will have an amino acid sequence having at least 75% amino acidsequence identity with the original humanized antibody amino acidsequences of either the heavy or the light chain more preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,and most preferably at least 95%, 98% or 99%. Identity or homology withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical with thehumanized anti-IL-23R residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence shall beconstrued as affecting sequence identity or homology.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, theantibody is an IgG antibody. Any isotype of IgG can be used, includingIgG₁, IgG₂, IgG₃, and IgG₄. Variants of the IgG isotypes are alsocontemplated. The humanized antibody may comprise sequences from morethan one class or isotype. Optimization of the necessary constant domainsequences to generate the desired biologic activity is readily achievedby screening the antibodies in the biological assays described in theExamples.

Likewise, either class of light chain can be used in the compositionsand methods herein. Specifically, kappa, lambda, or variants thereof areuseful in the present compositions and methods.

Any suitable portion of the CDR sequences from the non-human antibodycan be used. The CDR sequences can be mutagenized by substitution,insertion or deletion of at least one residue such that the CDR sequenceis distinct from the human and non-human antibody sequence employed. Itis contemplated that such mutations would be minimal. Typically, atleast 75% of the humanized antibody residues will correspond to those ofthe non-human CDR residues, more often 90%, and most preferably greaterthan 95%.

Any suitable portion of the FR sequences from the human antibody can beused. The FR sequences can be mutagenized by substitution, insertion ordeletion of at least one residue such that the FR sequence is distinctfrom the human and non-human antibody sequence employed. It iscontemplated that such mutations would be minimal. Typically, at least75% of the humanized antibody residues will correspond to those of thehuman FR residues, more often 90%, and most preferably greater than 95%,98% or 99%.

CDR and FR residues are determined according to the standard sequencedefinition of Kabat. Kabat et al. (1987) Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda Md. SEQID NOs: 1-5 show the heavy chain variable domain sequences of variousmouse and rat anti-human IL-23R antibodies, and SEQ ID NOs: 6-10 depictthe light chain variable domain sequences. SEQ ID NOs: 45-47 show theheavy chain variable domain sequences of various forms of humanizedmouse anti-human IL-23R clone 20D7 antibody, and SEQ ID NOs: 48-49 and53 depict the light chain variable domain sequences. FIGS. 1 and 2provide sequence lineups of heavy and light chain variable domains ofthe various antibodies of the present invention. CDRs are indicated inthe figures, and the individual CDR sequences are each presented withunique Sequence Identifiers (SEQ ID NOs: 11-40), as indicated in Tables2 and 4.

TABLE 2 Light Chain Sequences and Domains ANTIBODY SEQ ID V_(L) LIGHTCHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-L1 CDR-L2 CDR-L3 41F11 6 1-10824-34 50-56 89-97 8B10 7 1-108 24-34 50-56 89-97 3C11 8 1-108 24-3450-56 89-97 20D7 9 1-114 24-40 56-62  95-103 20E5 10 1-103 24-34 50-5689-97 hu20D7-IV 48 1-115 24-40 56-62  95-103 hu20D7-II-a 49 1-115 24-4056-62  95-103 hu20D7-II-b 53 1-115 24-40 56-62  95-103

TABLE 3 Heavy Chain Sequences and Domains ANTIBODY SEQ ID V_(H) HEAVYCHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-H1 CDR-H2 CDR-H3 41F11 1 1-11726-35 50-66 99-106 8B10 2 1-119 26-35 50-68 101-108  3C11 3 1-128 26-3752-67 100-117  20D7 4 1-120 26-35 50-66 99-109 20E5 5 1-119 26-35 50-6699-108 hu20D7-a 45 1-120 26-35 50-66 99-109 hu20D7-b 46 1-120 26-3550-66 99-109 hu20D7-c 47 1-120 26-35 50-66 99-109

In one embodiment, CDRs include variants of any single sequence CDRdisclosed herein (SEQ ID NOs: 11-40 and 52), in which the variantcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acidsubstitutions relative to the disclosed sequence, as determined usingTable 1.

Also contemplated are chimeric antibodies. As noted above, typicalchimeric antibodies comprise a portion of the heavy and/or light chainidentical with, or homologous to, corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity. See U.S. Pat. No. 4,816,567; and Morrisonet al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855.

Bispecific antibodies are also useful in the present methods andcompositions. As used herein, the term “bispecific antibody” refers toan antibody, typically a monoclonal antibody, having bindingspecificities for at least two different antigenic epitopes. In oneembodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs. See,e.g., Milstein et al. (1983) Nature 305: 537-39. Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan et al. (1985) Science 229:81. Bispecific antibodies includebispecific antibody fragments. See, e.g., Holliger et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:6444-48, Gruber et al. (1994) J. Immunol.152:5368.

In yet other embodiments, different constant domains may be appended tohumanized V_(L) and V_(H) regions derived from the CDRs provided herein.For example, if a particular intended use of an antibody (or fragment)of the present invention were to call for altered effector functions, aheavy chain constant domain other than IgG1 may be used. Although IgG1antibodies provide for long half-life and for effector functions, suchas complement activation and antibody-dependent cellular cytotoxicity,such activities may not be desirable for all uses of the antibody. Insuch instances an IgG4 constant domain, for example, may be used.

V. BIOLOGICAL ACTIVITY OF HUMANIZED ANTI-IL-23R ANTIBODIES

Antibodies having the characteristics identified herein as beingdesirable in a humanized anti-IL-23R antibody can be screened forinhibitory biologic activity in vitro or suitable binding affinity.Antagonist antibodies may be distinguished from agonist antibodies usingthe biological assays provided at Examples 5, 6 and 7. Antibodies thatexhibit agonist activity will not block the activity of IL-23, but willinstead stimulate the response typically caused by IL-23, such asincreasing cell proliferation in the Ba/F3 assay of Example 5 and/orincreasing IL-17 production in the splenocyte assay of Example 6.Although agonist antibodies may find use in some therapeuticindications, the anti-IL-23R antibodies disclosed herein are intended tobe antagonist antibodies unless otherwise indicated.

To screen for antibodies that bind to the epitope on human IL-23R boundby an antibody of interest (e.g., those that block binding of IL-23), aroutine cross-blocking assay such as that described in ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. Antibodies that bind to the same epitopeare likely to cross-block in such assays, but not all cross-blockingantibodies will necessarily bind at precisely the same epitope sincecross-blocking may result from steric hindrance of antibody binding byantibodies bind at overlapping epitopes, or even nearby non-overlappingepitopes.

Alternatively, epitope mapping, e.g., as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest. “Alanine scanningmutagenesis,” as described by Cunningham and Wells (1989) Science 244:1081-1085, or some other form of point mutagenesis of amino acidresidues in human IL-23R may also be used to determine the functionalepitope for an anti-IL-23R antibody of the present invention.Mutagenesis studies, however, may also reveal amino acid residues thatare crucial to the overall three-dimensional structure of IL-23R butthat are not directly involved in antibody-antigen contacts, and thusother methods may be necessary to confirm a functional epitopedetermined using this method.

The epitope bound by a specific antibody may also be determined byassessing binding of the antibody to peptides comprising fragments ofhuman IL-23R (SEQ ID NO: 41). A series of overlapping peptidesencompassing the sequence of IL-23R may be synthesized and screened forbinding, e.g. in a direct ELISA, a competitive ELISA (where the peptideis assessed for its ability to prevent binding of an antibody to IL-23Rbound to a well of a microtiter plate), or on a chip. Such peptidescreening methods may not be capable of detecting some discontinuousfunctional epitopes, i.e. functional epitopes that involve amino acidresidues that are not contiguous along the primary sequence of theIL-23R polypeptide chain.

The epitope bound by antibodies of the present invention may also bedetermined by structural methods, such as X-ray crystal structuredetermination (e.g., WO2005/044853), molecular modeling and nuclearmagnetic resonance (NMR) spectroscopy, including NMR determination ofthe H-D exchange rates of labile amide hydrogens in IL-23R when free andwhen bound in a complex with an antibody of interest (Zinn-Justin et al.(1992) Biochemistry 31:11335-11347; Zinn-Justin et al. (1993)Biochemistry 32:6884-6891).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J.Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLand preferably about 10 mg/mL to about 20 mg/mL. Crystallization may bebest achieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about4.0 to about 10.0, preferably about 7.0 to 8.5, e.g. 8.0. Specificbuffers useful in the precipitant solution may vary and are well-knownin the art. Scopes, Protein Purification: Principles and Practice, Thirded., (1994) Springer-Verlag, New York. Examples of useful buffersinclude, but are not limited to, HEPES, Tris, MES and acetate. Crystalsmay be grow at a wide range of temperatures, including 2° C., 4° C., 8°C. and 26° C.

Antibody:antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al. eds., Academic Press; U.S. Patent Application PublicationNo. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60;Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.;Roversi et al. (2000) Acta Cryst. D56:1313-1323).

Additional antibodies binding to the same epitope as an antibody of thepresent invention may be obtained, for example, by screening ofantibodies raised against IL-23R for binding to the epitope, or byimmunization of an animal with a peptide comprising a fragment of humanIL-23R comprising the epitope sequence. Antibodies that bind to the samefunctional epitope might be expected to exhibit similar biologicalactivities, such as blocking receptor binding, and such activities canbe confirmed by functional assays of the antibodies.

Antibody affinities may be determined using standard analysis. Preferredhumanized antibodies are those that bind human IL-23R with a K_(d) valueof no more than about 1×10⁻⁷ M; preferably no more than about 1×10⁻⁸ M;more preferably no more than about 1×10⁻⁹ M; and most preferably no morethan about 1×10⁻¹⁰ M or even 1×10⁻¹¹ M.

The antibodies and fragments thereof useful in the present compositionsand methods are biologically active antibodies and fragments. As usedherein, the term “biologically active” refers to an antibody or antibodyfragment that is capable of binding the desired the antigenic epitopeand directly or indirectly exerting a biologic effect. Typically, theseeffects result from the failure of IL-23 to bind IL-23 receptor. As usedherein, the term “specific” refers to the selective binding of theantibody to the target antigen epitope. Antibodies can be tested forspecificity of binding by comparing binding to IL-23R to binding toirrelevant antigen or antigen mixture under a given set of conditions.If the antibody binds to IL-23R at least 10, and preferably 50 timesmore than to irrelevant antigen or antigen mixture then it is consideredto be specific. An antibody that “specifically binds” to IL-23R does notbind to proteins that do not comprise the IL-23R-derived sequences, i.e.“specificity” as used herein relates to IL-23R specificity, and not anyother sequences that may be present in the protein in question. Forexample, as used herein, an antibody that “specifically binds” to apolypeptide comprising IL-23R will typically bind to FLAG®-hIL-23R,which is a fusion protein comprising IL-23R and a FLAG® peptide tag, butit does not bind to the FLAG® peptide tag alone or when it is fused to aprotein other than IL-23R.

IL-23R-specific binding compounds of the present invention, such asinhibitory IL-23R specific antibodies, can inhibit its biologicalactivity in any manner, including but not limited to reducing productionof IL-1β and TNF by peritoneal macrophages and IL-17 by T_(H)17 T cells.See Langrish et al. (2004) Immunol. Rev. 202:96-105. Anti-IL-23Rantibodies will also be able to inhibit the gene expression of IL-17A,IL-17F, CCL7, CCL17, CCL20, CCL22, CCR1, and GM-CSF. See Langrish et al.(2005) J. Exp. Med. 201:233-240. IL-23R-specific binding compounds ofthe present invention, such as anti IL-23R antibodies, will also blockthe ability of IL-23 to enhance proliferation or survival of T_(H)17cells. Cua and Kastelein (2006) Nat. Immunol. 7:557-559. The inhibitoryactivity of anti-IL-23R antibodies will be useful in the treatment ofinflammatory, autoimmune, and proliferative disorders. Examples of suchdisorders are described in PCT patent application publications WO04/081190; WO 04/071517; WO 00/53631; and WO 01/18051.

VI. PHARMACEUTICAL COMPOSITIONS

To prepare pharmaceutical or sterile compositions including IL-23Rantibody, the cytokine analogue or mutein, antibody thereto, or nucleicacid thereof, is admixed with a pharmaceutically acceptable carrier orexcipient. See, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa.(1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions or suspensions. See, e.g., Hardman et al. (2001) Goodman andGilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y.; Gennaro (2000) Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis et al.(eds.) (1993) Pharmaceutical Dosage Forms Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with an immunosuppressive agent,can be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio of LD₅₀ to ED₅₀. Antibodies exhibiting high therapeuticindices are preferred. The data obtained from these cell culture assaysand animal studies can be used in formulating a range of dosage for usein human. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration.

The mode of administration is not particularly important. Suitableroutes of administration may, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, intradermal, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections.Administration of antibody used in the pharmaceutical composition or topractice the method of the present invention can be carried out in avariety of conventional ways, such as oral ingestion, inhalation,topical application or cutaneous, subcutaneous, intraperitoneal,parenteral, intraarterial or intravenous injection.

Alternately, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto an arthritic joint or pathogen-induced lesion characterized byimmunopathology, often in a depot or sustained release formulation.Furthermore, one may administer the antibody in a targeted drug deliverysystem, for example, in a liposome coated with a tissue-specificantibody, targeting, for example, arthritic joint or pathogen-inducedlesion characterized by immunopathology. The liposomes will be targetedto and taken up selectively by the afflicted tissue.

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. Preferably,an administration regimen maximizes the amount of therapeutic deliveredto the patient consistent with an acceptable level of side effects.Accordingly, the amount of biologic delivered depends in part on theparticular entity and the severity of the condition being treated.Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available. See, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al. (2003)New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al.(2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J.Med. 343:1594-1602.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is substantially derived from the same species as the animaltargeted for treatment (e.g. a humanized antibody for treatment of humansubjects), thereby minimizing any immune response to the reagent.

Antibodies, antibody fragments, and cytokines can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, 1-7times per week, one week, two weeks, monthly, bimonthly, etc. Doses maybe provided, e.g., intravenously, subcutaneously, topically, orally,nasally, rectally, intramuscular, intracerebrally, intraspinally, or byinhalation. A preferred dose protocol is one involving the maximal doseor dose frequency that avoids significant undesirable side effects. Atotal weekly dose is generally at least 0.05 μg/kg, 0.2 μg/kg, 0.5μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10mg/kg, 25 mg/kg, 50 mg/kg body weight or more. See, e.g., Yang et al.(2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J.Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych.67:451-456; Portielji et al. (20003) Cancer Immunol. Immunother.52:133-144. The desired dose of a small molecule therapeutic, e.g., apeptide mimetic, natural product, or organic chemical, is about the sameas for an antibody or polypeptide, on a moles/kg basis.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with autoimmunedisease or pathogen-induced immunopathology and/or a reduction in theseverity of such symptoms that will or are expected to develop. Theterms further include ameliorating existing uncontrolled or unwantedautoimmune-related or pathogen-induced immunopathology symptoms,preventing additional symptoms, and ameliorating or preventing theunderlying causes of such symptoms. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with anautoimmune or pathogen-induced immunopathology disease or symptom, orwith the potential to develop such a disease or symptom.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of an IL-23R-specific bindingcompound, e.g. and antibody, that when administered alone or incombination with an additional therapeutic agent to a cell, tissue, orsubject is effective to prevent or ameliorate the autoimmune disease orpathogen-induced immunopathology associated disease or condition or theprogression of the disease. A therapeutically effective dose furtherrefers to that amount of the compound sufficient to result inamelioration of symptoms, e.g., treatment, healing, prevention oramelioration of the relevant medical condition, or an increase in rateof treatment, healing, prevention or amelioration of such conditions.When applied to an individual active ingredient administered alone, atherapeutically effective dose refers to that ingredient alone. Whenapplied to a combination, a therapeutically effective dose refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. An effective amount of therapeutic will decrease thesymptoms typically by at least 10%; usually by at least 20%; preferablyat least about 30%; more preferably at least 40%, and most preferably byat least 50%.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, antibody, steroid, chemotherapeutic agent,antibiotic, or radiation, are well known in the art, see, e.g., Hardmanet al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 10^(th) ed., McGraw-Hill, New York, N.Y.; Poole andPeterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: APractical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabnerand Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,Williams & Wilkins, Phila., PA. The pharmaceutical composition of theinvention may also contain other immunosuppressive or immunomodulatingagents. Any suitable immunosuppressive agent can be employed, includingbut not limited to anti-inflammatory agents, corticosteroids,cyclosporine, tacrolimus (i.e., FK-506), sirolimus, interferons, solublecytokine receptors (e.g., sTNRF and sIL-1R), agents that neutralizecytokine activity (e.g., inflixmab, etanercept), mycophenolate mofetil,15-deoxyspergualin, thalidomide, glatiramer, azathioprine, leflunomide,cyclophosphamide, methotrexate, and the like. The pharmaceuticalcomposition can also be employed with other therapeutic modalities suchas phototherapy and radiation.

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

VII. ANTIBODY PRODUCTION

In one embodiment, for recombinant production of the antibodies of thepresent invention, the nucleic acids encoding the two chains areisolated and inserted into one or more replicable vectors for furthercloning (amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.In one embodiment, both the light and heavy chains of a humanizedanti-IL-23R antibody of the present invention are expressed from thesame vector, e.g. a plasmid or an adenoviral vector.

Antibodies of the present invention may be produced by any method knownin the art. In one embodiment, antibodies are expressed in mammalian orinsect cells in culture, such as chinese hamster ovary (CHO) cells,human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells, babyhamster kidney (BHK) cells, Spodoptera frugiperda ovarian (Sf9) cells.In one embodiment, antibodies secreted from CHO cells are recovered andpurified by standard chromatographic methods, such as protein A, cationexchange, anion exchange, hydrophobic interaction, and hydroxyapatitechromatography. In one embodiment, resulting antibodies are concentratedand stored in 20 mM sodium acetate, pH 5.5.

In another embodiment, the antibodies of the present invention areproduced in yeast according to the methods described in WO2005/040395.Briefly, vectors encoding the individual light or heavy chains of anantibody of interest are introduced into different yeast haploid cells,e.g. different mating types of the yeast Pichia pastoris, which yeasthaploid cells are optionally complementary auxotrophs. The transformedhaploid yeast cells can then be mated or fused to give a diploid yeastcell capable of producing both the heavy and the light chains. Thediploid strain is then able to secret the fully assembled andbiologically active antibody. The relative expression levels of the twochains can be optimized, for example, by using vectors with differentcopy number, using transcriptional promoters of different strengths, orinducing expression from inducible promoters driving transcription ofthe genes encoding one or both chains.

In one embodiment, the respective heavy and light chains of a pluralityof different anti-IL-23R antibodies (the “original” antibodies) areintroduced into yeast haploid cells to create a library of haploid yeaststrains of one mating type expressing a plurality of light chains, and alibrary of haploid yeast strains of a different mating type expressing aplurality of heavy chains. These libraries of haploid strains can bemated (or fused as spheroplasts) to produce a series of diploid yeastcells expressing a combinatorial library of antibodies comprised of thevarious possible permutations of light and heavy chains. Thecombinatorial library of antibodies can then be screened to determinewhether any of the antibodies has properties that are superior (e.g.higher affinity for IL-23R) to those of the original antibodies. See.e.g., WO2005/040395.

In another embodiment, antibodies of the present invention are humandomain antibodies in which portions of an antibody variable domain arelinked in a polypeptide of molecular weight approximately 13 kDa. See,e.g., U.S. Pat. Publication No. 2004/0110941. Such single domain, lowmolecular weight agents provide numerous advantages in terms of ease ofsynthesis, stability, and route of administration.

VIII. USES

The present invention provides methods for using anti-IL-23R antibodiesand fragments thereof for the treatment and diagnosis of inflammatorydisorders and conditions, e.g., of the central nervous system,peripheral nervous system, and gastrointestinal tract, as well asautoimmune and proliferative disorders.

Methods are provided for the treatment of, e.g., multiple sclerosis(MS), including relapsing-remitting MS and primary progressive MS,Alzheimer's disease, amyotrophic lateral sclerosis (a.k.a. ALS; LouGehrig's disease), ischemic brain injury, prion diseases, andHIV-associated dementia. Also provided are methods for treatingneuropathic pain, posttraumatic neuropathies, Guillain-Barre syndrome(GBS), peripheral polyneuropathy, and nerve regeneration.

Provided are methods for treating or ameliorating one or more of thefollowing features, symptoms, aspects, manifestations, or signs ofmultiple sclerosis, or other inflammatory disorder or condition of thenervous system: brain lesions, myelin lesions, demyelination,demyelinated plaques, visual disturbance, loss of balance orcoordination, spasticity, sensory disturbances, incontinence, pain,weakness, fatigue, paralysis, cognitive impairment, bradyphrenia,diplopia, optic neuritis, paresthesia, gait ataxia, fatigue, Uhtoff'ssymptom, neuralgia, aphasia, apraxia, seizures, visual-field loss,dementia, extrapyramidal phenomena, depression, sense of well-being, orother emotional symptoms, chronic progressive myelopathy, and a symptomdetected by magnetic resonance imaging (MRI), includinggadolinium-enhancing lesions, evoked potential recordings, orexamination of cerebrospinal fluid. See, e.g., Kenealy et al. (2003) J.Neuroimmunol. 143:7-12; Noseworthy et al. (2000) New Engl. J. Med.343:938-952; Miller et al. (2003) New Engl. J. Med. 348:15-23; Chang etal. (2002) New Engl. J. Med. 346:165-173; Bruck and Stadelmann (2003)Neurol. Sci. 24 Suppl. 5:S265-S267.

Moreover, the present invention provides methods for treating anddiagnosing inflammatory bowel disorders, e.g., Crohn's disease,ulcerative colitis, celiac disease, and irritable bowel syndrome.Provided are methods for treating or ameliorating one or more of thefollowing symptoms, aspects, manifestations, or signs of an inflammatorybowel disorder: malabsorption of food, altered bowel motility,infection, fever, abdominal pain, diarrhea, rectal bleeding, weightloss, signs of malnutrition, perianal disease, abdominal mass, andgrowth failure, as well as intestinal complications such as stricture,fistulas, toxic megacolon, perforation, and cancer, and includingendoscopic findings, such as, friability, aphthous and linear ulcers,cobblestone appearance, pseudopolyps, and rectal involvement and, inaddition, anti-yeast antibodies. See, e.g., Podolsky, supra; Hanauer,supra; Horwitz and Fisher, supra.

Also contemplated is treatment of inflammatory disorders such aspsoriasis, atopic dermatitis, arthritis, including rheumatoid arthritis,osteoarthritis, and psoriatic arthritis, autoimmune disorders, such assystemic lupus erythematosus and type I diabetes, and proliferativedisorders such as cancer. See, e.g., PCT patent application publicationsWO 04/081190; WO 04/071517; WO 00/53631; and WO 01/18051.

The IL-23R binding compounds of the present invention can also be usedin combination with one or more antagonists of other cytokines (e.g.antibodies), including but not limited to, IL-23p19, IL-17A, IL-17F,TNF-α, IL-1β, IL-6 and TGF-β. See, e.g., Veldhoen (2006) Immunity24:179-189; Dong (2006) Nat. Rev. Immunol. 6(4):329-333. In variousembodiments, an IL-23R binding compound of the invention is administeredbefore, concurrently with, or after administration of the anotherantagonist or antagonists, such as an anti-IL-17A antibody. In oneembodiment, an IL-17A binding compound is used in treatment of the acuteearly phase of an adverse immune response (e.g. MS, Crohn's Disease)alone or in combination with an IL-23R antagonist antibody of thepresent invention. In the latter case, the IL-17A binding compound maybe gradually decreased and treatment with the antagonist of IL-23R aloneis continued to maintain suppression of the adverse response.Alternatively, antagonists to IL-23p19, IL-1β, IL-6 and/or TGF-β may beadministered concurrently, before or after an IL-23R binding compound ofthe present invention. See Cua and Kastelein (2006) Nat. Immunol.7:557-559; Tato and O'Shea (2006) Nature 441:166-168; Iwakura andIshigame (2006) J. Clin. Invest. 116:1218-1222.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments. The specific embodiments described hereinare offered by way of example only, and the invention is to be limitedby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

EXAMPLES Example 1 General Methods

Standard methods in molecular biology are described. Maniatis et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif. Standard methods also appear in Ausbel et al.(2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley andSons, Inc. New York, N.Y., which describes cloning in bacterial cellsand DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed. Coligan et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemicalmodification, post-translational modification, production of fusionproteins, glycosylation of proteins are described. See, e.g., Coligan etal. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley andSons, Inc., New York; Ausubel et al. (2001) Current Protocols inMolecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life ScienceResearch, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001)BioDirectory, Piscataway, N.J., pp. 384-391. Production, purification,and fragmentation of polyclonal and monoclonal antibodies are described.Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, JohnWiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlowand Lane, supra. Standard techniques for characterizing ligand/receptorinteractions are available. See, e.g., Coligan et al. (2001) CurrentProtocols in Immunology, Vol. 4, John Wiley, Inc., New York.

Methods for flow cytometry, including fluorescence activated cellsorting detection systems (FACS®), are available. See, e.g., Owens etal. (1994) Flow Cytometry Principles for Clinical Laboratory Practice,John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd)ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry,John Wiley and Sons, Hoboken, N.J. Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable. Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

Standard methods of histology of the immune system are described. See,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available. See, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16: 741-742;Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren etal. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690.

Example 2 Generation and Humanization of Anti-Human IL-23R Antibodies

Anti-human IL-23R antibodies are generated by immunizing rats or micewith the extracellular domain of human-IL-23R (residues 24-353 of SEQ IDNO: 41), either with a C-terminal histidine tag or as an Ig (human IgG1Fc) fusion protein. Monoclonal antibodies are then prepared by standardmethods.

The humanization of antibodies is described generally, e.g., in PCTpatent application publications WO 2005/047324 and WO 2005/047326.Exemplary humanized heavy- and light-chain variable domain sequences areprovided at FIGS. 1 and 2, respectively, and in the Sequence Listing.

Briefly, the amino acid sequence of the non-human VH domain (e.g. SEQ IDNOs: 1-5) is compared to a group of five human VH germline amino acidsequences; one representative from subgroups IGHV1 and IGHV4 and threerepresentatives from subgroup IGHV3. The VH subgroups are listed inM.-P. Lefranc (2001) “Nomenclature of the Human Immunoglobulin Heavy(IGH) Genes”, Experimental and Clinical Immunogenetics 18:100-116. Theframework sequences of the human germline sequence with the closestmatch are used to construct a humanized VH domain.

The rodent anti-huIL-23R antibodies disclosed herein are all of thekappa subclass of VL. The amino acid sequences of the non-human VLdomain (e.g. SEQ ID NOs: 6-10) is compared to a group of four human VLkappa germline amino acid sequences. The group of four is comprised ofone representative from each of four established human VL subgroupslisted in V. Barbie & M.-P. Lefranc (1998) “The Human ImmunoglobulinKappa Variable (IGKV) Genes and Joining (IGKJ) Segments”, Experimentaland Clinical Immunogenetics 15:171-183 and M.-P. Lefranc (2001)“Nomenclature of the Human Immunoglobulin Kappa (IGK) Genes”,Experimental and Clinical Immunogenetics 18:161-174. The four subgroupsalso correspond to the four subgroups listed in Kabat et al. (1991-5thEd.) “Sequences of Proteins of Immunological Interest”, U.S. Departmentof Health and Human Services, NIH Pub. 91-3242, pp. 103-130. Theframework sequences of the human germline sequence with the closestmatch are used to construct a humanized VL domain.

Based on the foregoing analysis, a humanized form of mouse antibodyclone 20D7 is constructed using human heavy chain sequence from subgroupI (germline sequence DP-14). The sequence of the resulting humanizedheavy chain variable domain (hu20D7-a) is provided at SEQ ID NO: 45. Avariant of hu20D7-a, hu20D7-b (SEQ ID NO: 46), is identical except for aT72L substitution. A second variant, hu20D7-c (SEQ ID NO: 47), isidentical to hu20D7-b except for an M70F substitution. Both of thesesequence variations are in the framework region between CDRH2 and CDRH3,and are illustrated as bold type-face residues in FIG. 1. A full-lengthhumanized heavy chain, comprising the hu20D7-c variable domain and ahuman IgG1 constant domain, is provided at SEQ ID NO: 50.

Light chain variable domain sequences are also constructed from mouseantibody clone 20D7. One light chain variable domain sequence, hu20D7-IV(SEQ ID NO: 48), comprises the CDR sequences of the parental mouseantibody and human light chain framework sequences from light chainsubgroup IV (germline sequence Z-B3). Another light chain variabledomain sequence, hu20D7-II-a (SEQ ID NO: 49), comprises the CDRsequences of the parental mouse antibody and human light chain frameworksequences from light chain subgroup II (germline sequence Z-A19). Anadditional light chain variable domain sequence variant (hu20D7-II-b) isprovided at SEQ ID NO:53, in which the serine residue at position 32 isreplaced by threonine (S32T). The serine is replaced to reduce thelikelihood of deamidation of the preceding asparagine residue. SeeBischoff & Kolbe (1994) J. Chromatog. 662:261. All three light chainvariable domain sequences are presented in FIG. 2. Full-length humanizedlight chains, comprising the hu20D7-II-a and hu20D7-II-b variabledomains in the context of human kappa light chains, are provided at SEQID NOs: 51 and 54, respectively.

Any pairwise combination of the humanized heavy and light chainsdescribed in this example (e.g. the hu20D7-II-b light chain and thehu20D7-c heavy chain) can be used to create a functionalanti-human-IL-23R antibody. Full-length heavy and light chains can beconstructed from any of the variable domains disclosed herein by analogywith the construction of SEQ ID NOs: 50 and 51 from SEQ ID NOs: 47 and49, respectively. Alternatively, other heavy chain constant domains(e.g. IgG₂) or other light chains (e.g. lambda) may be constructed usingknown human immunoglobulin sequences.

Once the target amino acid sequences of the variable heavy and lightchains are determined, plasmids encoding the full-length humanizedantibody may be generated. Plasmid sequences may be altered using Kunkelmutagenesis (see, e.g., Kunkel T A. (1985) Proc. Natl. Acad. Sci. U.S.A.82:488-492) to change the DNA sequence to the target humanized antibodysequences. Simultaneously, codon optimization may be performed toprovide for potentially optimal expression. Sequence optimization mayalso be performed by commercial vendors, such as GENEART, Inc., Toronto,ON, Canada.

In the case of the humanization of the 20D7 antibody, simplesubstitution of the best-match human germline frameworks into the rodentvariable domains produced antibodies (e.g. SEQ ID NO: 45 paired with SEQID NO: 48 or 49) with substantially reduced binding affinity for IL-23Rwhen compared with the parental (rodent) forms. To determine the sourceof the reduced affinity, hemichimeric constructs were created in whichthe humanized light and heavy chains were paired with chimeric heavy andlight chains, respectively. Chimeric chains include the rodent variabledomain and a human constant domain. It had been previously determinedearlier that chimeric forms of the 20D7 antibodies retained essentiallythe same affinity as the parental rodent antibody. It was observed thatthe hemichimeric antibody with the humanized heavy chain had reducedbinding, whereas the hemichimeric antibody with the humanized lightchain retained the affinity of the parental antibody, indicating thatthe problem was with the humanized heavy chain. The humanized heavychain was then scrutinized to find positions at which the humanizedframework sequences differed from the parental (rodent) frameworksequences, which positions were considered likely causes of the reducedaffinity. Sequence variants were produced at several positions, andheavy chains having T72L and M70F substitutions were found tosubstantially improve binding affinity. See Example 4, infra. See, e.g.,Tramontano et al. (1990) J. Mol. Biol. 215:175. (Note that unlessotherwise indicated, sequence numbering in this patent applicationrelates to the numbering of the sequence listing, and not to alternativenumbering schemes such as the Kabat system.)

A humanized form of rat antibody clone 8B10 is constructed in a similarfashion, using human heavy chain sequence from subgroup III (germlinesequence DP-46). The sequence of the resulting humanized heavy chainvariable domain (hu8B10) is provided at SEQ ID NO: 57. Variants ofhu8B10 heavy chain variable domain may be created to improve bindingaffinity or otherwise improve the properties of the resulting antibody.For example, the asparagine residue at position 30 of SEQ ID NO: 57 (inCDRH1) may be changed to threonine (N30T) to improve binding affinity.Framework residues may also be altered, such as residues A24, E46, W47,N76, N79, L81, or R100. Exemplary framework changes include E46D andW47L.

Light chain variable domain sequences are also constructed from ratantibody clone 8B10. A hu8B10 light chain variable domain sequencecomprising the CDR sequences of the parental rat antibody and humanlight chain framework sequences from light chain subgroup kappa-1(germline sequence Z-012) is provided at SEQ ID NO: 58.

Example 3 Determining the Equilibrium Dissociation Constant (K_(d)) forAnti-Human IL-23R Antibodies Using KinExA Technology

The equilibrium dissociation constants (K_(d)) for anti human IL-23Rantibodies are determined using the KinExA 3000 instrument. SapidyneInstruments Inc., Boise Id., USA. KinExA uses the principle of theKinetic Exclusion Assay method based on measuring the concentration ofuncomplexed antibody in a mixture of antibody, antigen andantibody-antigen complex. The concentration of free antibody is measuredby exposing the mixture to a solid-phase immobilized antigen for a verybrief period of time. In practice, this is accomplished by flowing thesolution phase antigen-antibody mixture past antigen-coated particlestrapped in a flow cell. Data generated by the instrument are analyzedusing custom software. Equilibrium constants are calculated using amathematical theory based on the following assumptions:

1. The binding follows the reversible binding equation for equilibrium:

k_(on)[Ab][Ag]=k_(off)[AbAg]

2. Antibody and antigen bind 1:1 and total antibody equalsantigen-antibody complex plus free antibody.

3. Instrument signal is linearly related to free antibody concentration.

PMMA particles (Sapidyne, Cat No. 440198) are coated with biotinylatedIL-23R (or a fragment thereof, such as the extracellular domain)according to Sapidyne “Protocol for coating PMMA particles withbiotinylated ligands having short or nonexistent linker arms.” EZ-linkTFP PEO-biotin (Pierce, Cat. No. 21219) is used to make biotinylatedIL-23R, as per the manufacturer's recommendations (Pierce bulletin0874).

Antibody r8B10 showed a K_(d) of 4.2×10⁻¹¹ M, and antibody m20D7 showeda K_(d) of 9×10⁻¹² M, for binding to human IL-23R as determined byKinExA analysis.

Example 4 Determining the Equilibrium Dissociation Constant (K_(d)) forHumanized Anti-Human IL-23R Antibodies Using BIAcore® Label-FreeInteraction Analysis System Technology

BIAcore® label-free interaction analysis system determinations areperformed essentially as described at Example 4 of U.S. PatentApplication Publication No. 2007/0048315. Briefly, ligands (anti-IL-23RmAbs) are immobilized on a BIAcore® label-free interaction analysissystem CM5 sensor chip using standard amine-coupling procedure. Kineticconstants for the various interactions are determined usingBIAevaluation software 3.1. The K_(d) is determined using the calculateddissociation and association rate constants.

BIAcore® label-free interaction analysis system determinations of theK_(d) for the mouse 20D7 antibody for human IL-23R showed thatreplacement of mouse framework sequences with human framework sequences(hu20D7-a, SEQ ID NO: 45) reduced binding affinity by approximately50-fold, from approximately 85 pM to approximately 4300 pM. Introductionof the T72L mutation (hu20D7-b, SEQ ID NO: 46) into the heavy chainimproved the K_(d) to approximately 360 pM, and introduction of the T72Lalong with the M70F mutation (hu20D7-c, SEQ ID NO: 47) further improvedthe K_(d) to approximately 230 pM. The mutations introduced in thehu20D7-c heavy chain thus represent an improvement of nearly 20-fold inbinding affinity compared to the simple humanization without subsequentoptimization.

By way of comparison, the parental rat 8B10 antibody exhibited a K_(d)of approximately 300 pM for human IL-23R when assessed using the sameassay.

Example 5 Proliferation Bioassays for the Assessment of NeutralizingAnti-IL-23R Antibodies

The ability of a monoclonal antibody to biologically neutralizeIL-23/IL-23R is assessed by the application of short-term proliferationbioassays that employ cells that express recombinant IL-23 receptors.The transfectant Ba/F3-2.2lo cells proliferate in response to humanIL-23 and the response can be inhibited by a neutralizing anti-IL-23Rantibody. The concentration of IL-23 chosen for the assay is selected tobe within the linear region of the dose-response curve, near plateau andabove EC50. Proliferation, or lack thereof, is measured by colorimetricmeans using Alamar Blue, a growth indicator dye based on detection ofmetabolic activity. The ability of an antibody to neutralizeIL-23/IL-23R is assessed by its IC50 value, or concentration of antibodythat induces half-maximal inhibition of IL-23 proliferation.

The assay is performed essentially as follows. Ba/F3 transfectants aremaintained in RPMI-1640 medium, 10% fetal calf serum, 50 μM2-mercaptoethanol, 2 mM L-Glutamine, 50 μg/mL penicillin-streptomycin,and 10 ng/mL mouse IL-3. Proliferation bioassays are performed inRPMI-1640 medium, 10% fetal calf serum, 50 μM 2-mercaptoethanol, 2 mML-Glutamine, and 50 μg/mL penicillin-streptomycin.

Assays are performed in 96-well flat bottom plates (Falcon 3072 orsimilar) in 150 μL per well. Both IL-23 and anti-IL-23R antibodies areadded at a series of concentrations, e.g. 1:3 serial dilutions.Titrations of the anti-IL-23R antibody of interest are optionallypre-incubated with cells prior to addition of IL-23. Bioassay plates areincubated in a humidified tissue culture chamber (37 C, 5% CO₂) for40-48 hr. At the end of the culture time, Alamar Blue (Biosource Cat#DAL1100) is added at 16.5 μL/well and allowed to develop for 5-12hours. Absorbance is then read at 570 nm and 600 nm (VERSAmax MicroplateReader, Molecular Probes, Eugene, Oreg., USA), and an OD₅₇₀₋₆₀₀ isobtained. Duplicates are run for each sample. Absorbance is plottedagainst cytokine or antibody concentration using GraphPad Prism® 3.0software (Graphpad Software Inc., San Diego, Calif., USA), and IC50values are determined using non-linear regression (curve fit) ofsigmoidal dose-response.

Example 6 Mouse Splenocyte Assay for IL-23 Based on IL-17 Production

The biological activity of anti-IL-23R antibodies of the presentinvention may be assessed using the mouse splenocyte assay essentiallyas described in Aggarwal et al. (2003) J. Biol. Chem. 278:1910 andStumhofer et al. (2006) Nature Immunol. 7:937. The mouse splenocyteassay measures the activity of IL-23 in a sample as a level of IL-17production by murine splenocytes. The inhibitory activity of anti-IL-23Rantibodies is then assessed by determining the concentration of antibodynecessary to reduce the IL-23/IL-23R activity in a given sample by 50%(the IC50). The IC50 as measured by this assay is greater than or equalto the equilibrium dissociation binding constant (K_(d)), i.e. the K_(d)may be equal to or lower than the IC50. As always, lower IC50 and K_(d)values reflect higher activities and affinities.

Briefly, spleens are obtained from 8-12 wk old female C57BL/6J mice(Jackson Laboratories, Bar Harbor, Me., USA). Spleens are ground,pelleted twice, and filtered through a cell strainer (70 μm nylon). Therecovered cells are cultured in 96-well plates (4×10⁵ cells/well) in thepresence of human IL-23 (10 ng/ml, ˜170 pM) and mouse-anti-CD3eantibodies (1 μg/ml) (BD Pharmingen, Franklin Lakes, N.J., USA), with orwithout the anti-IL-23R antibody to be assayed. Anti IL-23R antibodiesare added at a series of 3-fold dilutions. Cells are cultured for 72hours, pelleted, and the supernatant is assayed for IL-17 levels bysandwich ELISA.

IL-17 ELISA is performed as follows. Plates are coated with a captureanti-IL-17 antibody (100 ng/well) overnight at 4° C., washed andblocked. Samples and standards are added and incubated for two hours atroom temperature with shaking Plates are washed, and a biotinylatedanti-IL-17 detection antibody (100 ng/well) is added and incubated forone hour at room temperature with shaking. The capture and detectionantibodies are different antibodies that both bind to mouse IL-17 but donot cross-block. Plates are washed, and bound detection antibody isdetected using streptavidin-HRP (horseradish peroxidase) and TMB(3,3′,5,5′-tetramethylbenzidine). The plate is then read at 450-650 nmand the concentration of IL-17 in samples is calculated by comparisonwith standards.

Example 7 Characterization of Anti-IL-23R Antibody Hum20D7

A humanized anti-IL-23R antibody is generated using the heavy chainsequence hu20D7-c and the light chain sequence hu20D7-II-b, for whichsequences are provided at SEQ ID NOs: 50 and 54, respectively. Theresulting antibody is referred to in this example as hum20D7. Theantibody is prepared from mammalian cells using a vector harboring DNAsequences encoding the heavy and light chains, as provided at SEQ IDNOs: 55 (hu20D7-c) and 56 (hu20D7-II-b), respectively, although otherDNA sequences encoding the same polypeptide sequences may also be used.

Hum20D7 has a K_(d) of 131 pM for human IL-23R when assayed by BIAcoreanalysis, essentially as described in Example 4 (supra).

The biological activity of hum20D7 is also assessed using a humansplenocyte assay, essentially as described in Example 6 (supra) with theexception that splenocytes are obtained from human spleens rather thanmouse, no anti-CD3e antibody is used, and that IFN-γ is the readoutrather than IL-17. The assay measures the activity of IL-23 in a sampleby determining the level of IFN-γ production by human primarysplenocytes. Human splenocytes are exposed to human IL-23 (170 pM) inthe presence of various concentrations of anti-IL-23R antibody hum20D7,or in the absence of the antibody. IFN-γ is detected by sandwich ELISA.Hum20D7 exhibits an IC50 of 34 pM in the human splenocyte assay.

The biological activity of hum20D7 is further assessed using a KIT225STAT-3 phosphorylation assay, essentially as described in Parham et al.(2002) J. Immunol. 168:5699. Human KIT225 cells, a leukemic T cell line,are stimulated with 138 pM human IL-23 in the presence of variousconcentrations of anti-IL-23R antibody hum20D7, or in the absence of theantibody. IL-23 activity is measured by detecting the level of STAT3phosphorylation. Hum20D7 exhibits an IC50 of 34 pM in the KIT225 assay.

Table 4 provides a brief description of the sequences in the sequencelisting.

TABLE 4 Sequence Identifiers SEQ ID NO: Description 1 41F11 Heavy ChainVariable 2 8B10 Heavy Chain Variable 3 3C11 Heavy Chain Variable 4 20D7Heavy Chain Variable 5 20E5 Heavy Chain Variable 6 41F11 Light ChainVariable 7 8B10 Light Chain Variable 8 3C11 Light Chain Variable 9 20D7Light Chain Variable 10 20E5 Light Chain Variable 11 41F11 CDRH1 12 8B10CDRH1 13 3C11 CDRH1 14 20D7 CDRH1 15 20E5 CDRH1 16 41F11 CDRH2 17 8B10CDRH2 18 3C11 CDRH2 19 20D7 CDRH2 20 20E5 CDRH2 21 41F11 CDRH3 22 8B10CDRH3 23 3C11 CDRH3 24 20D7 CDRH3 25 20E5 CDRH3 26 41F11 CDRL1 27 8B10CDRL1 28 3C11 CDRL1 29 20D7 CDRL1 30 20E5 CDRL1 31 41F11 CDRL2 32 8B10CDRL2 33 3C11 CDRL2 34 20D7 CDRL2 35 20E5 CDRL2 36 41F11 CDRL3 37 8B10CDRL3 38 3C11 CDRL3 39 20D7 CDRL3 40 20E5 CDRL3 41 Human IL-23R 42Murine IL-23R 43 Human IL-12Rβ1 44 Murine IL-12Rβ1 45 hu20D7-a HeavyChain Variable 46 hu20D7-b Heavy Chain Variable 47 hu20D7-c Heavy ChainVariable 48 hu20D7-IV Light Chain Variable 49 hu20D7-II-a Light ChainVariable 50 hu20D7-c Heavy Chain 51 hu20D7-II-a Light Chain 52hu20D7-II-b CDRL1 53 hu20D7-II-b Light Chain Variable 54 hu20D7-II-bLight Chain 55 hu20D7-c Heavy Chain DNA 56 hu20D7-II-b Light Chain DNA57 hu8B10 Heavy Chain Variable 58 hu8B10 Light Chain Variable

1. A binding compound that binds to human IL-23R comprising: a) anantibody light chain variable domain, or antigen binding fragmentthereof, comprising one or more CDR sequences selected from the groupconsisting of SEQ ID NOs: 26-40 and 52; and b) an antibody heavy chainvariable domain, or antigen binding fragment thereof, comprising one ormore CDR sequences selected from the group consisting of SEQ ID NOs:11-25. 2-6. (canceled)
 7. The binding compound of claim 1 comprising: a)an antibody light chain variable domain, or antigen binding fragmentthereof, comprising three CDR sequences selected from the groupconsisting of SEQ ID NOs: 26-40 and 52; and b) an antibody heavy chainvariable domain, or antigen binding fragment thereof, comprising threeCDR sequences selected from the group consisting of SEQ ID NOs: 11-25.8-9. (canceled)
 10. The binding compound of claim 1 comprising: a) lightchain variable domain, or antigen binding fragment thereof, comprising:at least one CDRL1 from the group consisting of SEQ ID NOs: 26-30 and52; at least one CDRL2 from the group consisting of SEQ ID NOs: 31-35;and at least one CDRL3 from the group consisting of SEQ ID NOs: 36-40;and b) a heavy chain variable domain, or antigen binding fragmentthereof, comprising: at least one CDRH1 from the group consisting of SEQID NOs: 11-15; at least one CDRH2 from the group consisting of SEQ IDNOs: 16-20; and at least one CDRH3 from the group consisting of SEQ IDNOs: 21-25. 11-12. (canceled)
 13. A binding compound that binds to humanIL-23R, comprising: a) a light chain variable domain, or antigen bindingfragment thereof, comprising CDRL1, CDRL2 and CDRL3, wherein: CDRL1comprises the sequence of SEQ ID NO: 29 or 52 or a variant thereof;CDRL2 comprises the sequence of SEQ ID NO: 34 or a variant thereof; andCDRL3 comprises the sequence of SEQ ID NO: 39 or a variant thereof; andb) a heavy chain variable domain, or antigen binding fragment thereof,comprising CDRH1, CDRH2 and CDRH3, wherein: CDRH1 comprises the sequenceof SEQ ID NO: 14 or a variant thereof; CDRH2 comprises the sequence ofSEQ ID NO: 19 or a variant thereof; and CDRH3 comprises the sequence ofSEQ ID NO: 24 or a variant thereof; wherein each variant comprises up tofive conservatively modified amino acid substitutions.
 14. The bindingcompound of claim 13 wherein: a) the light chain variable domain, orantigen binding fragment thereof, comprises a sequence selected from thegroup consisting of SEQ ID NOs: 48, 49 and 53; and b) the heavy chainvariable domain, or antigen binding fragment thereof, comprises asequence selected from the group consisting of SEQ ID NOs: 45, 46 and47. 15-16. (canceled)
 17. A binding compound that binds to human IL-23Rcomprising: a) an antibody light chain variable domain, or antigenbinding fragment thereof, comprising CDRL1, CDRL2 and CDRL3, wherein:CDRL1 comprises the sequence of SEQ ID NO: 29 or 52; CDRL2 comprises thesequence of SEQ ID NO: 34; and CDRL3 comprises the sequence of SEQ IDNO: 39; and b) a heavy chain variable domain, or antigen bindingfragment thereof, comprising CDRH1, CDRH2 and CDRH3, wherein: CDRH1comprises the sequence of SEQ ID NO: 14; CDRH2 comprises the sequence ofSEQ ID NO: 19; and CDRH3 comprises the sequence of SEQ ID NO:
 24. 18-19.(canceled)
 20. A binding compound that binds to human IL-23R,comprising: a) an antibody light chain variable domain, or antigenbinding fragment thereof, consisting essentially of a sequence selectedfrom the group consisting of SEQ ID NOs: 48, 49 and 53; and b) anantibody heavy chain variable domain, or antigen binding fragmentthereof, consisting essentially of a sequence selected from the groupconsisting of SEQ ID NOs: 45, 46 and
 47. 21. (canceled)
 22. An antibody,or antigen binding fragment thereof, that is able to block the bindingof a binding compound comprising: a) an antibody light chain variabledomain comprising the sequence of SEQ ID NO: 54; and b) an antibodyheavy chain variable domain comprising the sequence of SEQ ID NO: 50, tohuman IL-23R in a cross-blocking assay.
 23. An isolated nucleic acidencoding at least one of the light chain variable domain or heavy chainvariable domain of the binding compound of claim
 17. 24. An expressionvector comprising the nucleic acid of claim 23 operably linked tocontrol sequences that are recognized by a host cell when the host cellis transfected with the vector.
 25. A host cell comprising theexpression vector of claim
 24. 26. A method of producing a polypeptidecomprising: culturing the host cell of claim 25 in culture medium underconditions wherein the nucleic acid sequence is expressed, therebyproducing polypeptides comprising the light and heavy chain variabledomains; and recovering the polypeptides from the host cell or culturemedium.
 27. The binding compound of claim 17, further comprising: a)human germline light chain framework sequences in the antibody lightchain variable domain; and b) human germline heavy chain frameworksequences in the antibody heavy chain variable domain.
 28. The bindingcompound of claim 17, further comprising a heavy chain constant regioncomprising a γ1 human heavy chain constant region or a variant thereof,wherein the constant region variant comprises up to 20 conservativelymodified amino acid substitutions.
 29. The binding compound of claim 17,further comprising a heavy chain constant region comprising a γ4 humanheavy chain constant region or a variant thereof, wherein the constantregion variant comprises up to 20 conservatively modified amino acidsubstitutions.
 30. The binding compound of claim 17, wherein the bindingcompound is an antibody fragment selected from the group consisting ofFab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, and a diabody.
 31. A method ofsuppressing an immune response in a human subject comprisingadministering to a subject in need thereof a binding compound of claim17, or a antigen binding fragment thereof, in an amount effective toblock the biological activity of IL-23R.
 32. The method of claim 31,wherein the biological activity is an inflammatory response.
 33. Themethod of claim 32, wherein the subject has a disorder selected from thegroup consisting of arthritis, psoriasis and inflammatory bowel disease.34. The method of claim 31, wherein the biological activity is anautoimmune response.
 35. The method of claim 34, wherein the subject hasa disorder selected from the group consisting of multiple sclerosis,systemic lupus erythematosus and diabetes.
 36. The method of claim 31,wherein the subject has cancer and the biological activity is a Th17response.
 37. (canceled)
 38. A pharmaceutical composition comprising thebinding compound of claim 17 in combination with a pharmaceuticallyacceptable carrier or diluent.
 39. (canceled)
 40. An antibody, orantigen binding fragment thereof, that is able to block binding of humanIL-23R to the antibody deposited with ATCC under accession numberPTA-7800 in a cross-blocking assay.
 41. An antibody, or antigen bindingfragment thereof, that is able to block binding of human IL-23R to theantibody deposited with ATCC under accession number PTA-7801 in across-blocking assay.
 42. The binding compound of claim 14 wherein: a)the light chain variable domain, or antigen binding fragment thereof,comprises the sequence of SEQ ID NO: 53, and b) the heavy chain variabledomain, or antigen binding fragment thereof, comprises the sequence ofSEQ ID NO:
 47. 43. The binding compound of claim 42 comprising: a) anantibody light chain variable domain, or antigen binding fragmentthereof, comprising the sequence of SEQ ID NO: 54; and b) an antibodyheavy chain variable domain, or antigen binding fragment thereof,comprising the sequence of SEQ ID NO:
 50. 44. A binding compound thatbinds to human IL-23R, comprising: a) an antibody light chain variabledomain, or antigen binding fragment thereof, comprising the sequence ofSEQ ID NO: 58; and b) an antibody heavy chain variable domain, orantigen binding fragment thereof, comprising the sequence of SEQ ID NO:57.